Systems and Methods for Enhanced Nucleic acid Seperation

ABSTRACT

Methods and apparatus for separating, concentrating and/or detecting molecules based on differences in binding affinity to a probe are provided. The molecules may be differentially modified. The molecules may be differentially methylated nucleic acids. The methods can be used in fields such as epigenetics or oncology to selectively concentrate or detect the presence of specific biomolecules or differentially modified biomolecules, to provide diagnostics for disorders such as fetal genetic disorders, to detect biomarkers in cancer, organ failure, disease states, infection or the like.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. application Ser. No.61/488,585 filed 20 May 2011 entitled SYSTEMS AND METHODS FOR ENHANCEDSCODA, the entirety of which is hereby incorporated by reference.

FIELD OF THE INVENTION

Embodiments of the present invention relate to the induced movement ofparticles such as nucleic acids, proteins and other molecules throughmedia such as gels and other matrices. Some embodiments provide methodsand apparatus for selectively purifying, separating, concentratingand/or detecting particles of interest. Some embodiments provide methodsand apparatus for selectively purifying, separating, concentratingand/or detecting differentially modified particles of interest. Someembodiments provide methods and apparatus for selectively purifying,separating, concentrating and/or detecting differentially methylatedDNA. Some embodiments are used in fields such as epigenetics, oncology,or various fields of medicine. Some embodiments are used to detect fetalgenetic disorders, biomarkers indicative of cancer or a risk of cancer,organ failure, disease states, infections, or the like.

BACKGROUND

One mechanism for purifying, separating, or concentrating molecules ofinterest is called Synchronous Coefficient Of Drag Alteration (or“SCODA”) based purification. SCODA, known in some embodiments asscodaphoresis, is an approach that may be applied for purifying,separating, or concentrating particles. SCODA may be applied, forexample, to DNA, RNA and other molecules including proteins andpolypeptides.

SCODA based transport is used to produce net motion of a molecule ofinterest by synchronizing a time-varying driving force, which wouldotherwise impart zero net motion, with a time-varying drag (or mobility)alteration. If application of the driving force and periodic mobilityalteration are appropriately coordinated, the result is net motiondespite zero time-averaged forcing. With careful choice of both thetemporal and spatial configuration of the driving and mobility alteringfields, unique velocity fields can be generated, in particular avelocity field that has a non-zero divergence, such that this method oftransport can be used for separation, purification and/or concentrationof particles.

SCODA is described in the following publications:

-   -   U.S. Patent Publication No. 2009/0139867 entitled “Scodaphoresis        and methods and apparatus for moving and concentrating        particles”;    -   PCT Publication No. WO 2006/081691 entitled “Apparatus and        methods for concentrating and separating particles such as        molecules”;    -   PCT Publication No. WO 2009/094772 entitled “Methods and        apparatus for particle introduction and recovery”;    -   PCT Publication No. WO 2009/001648 entitled “Systems and methods        for enhanced SCODA”    -   PCT Publication No. WO 2010/051649 entitled “Systems and methods        for enhanced SCODA”;    -   PCT Publication No. WO 2010/121381 entitled “System and methods        for detection of particles”;    -   Marziali, A.; Pel, J.; Bizotto, D.; Whitehead, L. A., “Novel        electrophoresis mechanism based on synchronous alternating drag        perturbation”, Electrophoresis 2005, 26, 82-89;    -   Broemeling, D.; Pel, J.; Gunn, D.; Mai, L.; Thompson, J.; Poon,        H.; Marziali, A., “An Instrument for Automated Purification of        Nucleic Acids from Contaminated Forensic Samples”, JALA 2008,        13, 40-48;    -   Pel, J.; Broemeling, D.; Mai, L.; Poon, H.; Tropini, G.; Warren,        R.; Holt, R.; Marziali, A., “Nonlinear electrophoretic response        yields a unique parameter for separation of biomolecules”, PNAS        2008, vol. 106, no. 35, 14796-14801; and    -   So, A.; Pel, J.; Rajan, S.; Marziali, A., “Efficient genomic DNA        extraction from low target concentration bacterial cultures        using SCODA DNA extraction technology”, Cold Spring Harb Protoc        2010, 1150-1153,        each of which is incorporated herein by reference.

SCODA can involve providing a time-varying driving field component thatapplies forces to particles in some medium in combination with atime-varying mobility-altering field component that affects the mobilityof the particles in the medium. The mobility-altering field component iscorrelated with the driving field component so as to provide atime-averaged net motion of the particles. SCODA may be applied to causeselected particles to move toward a focus area.

In one embodiment of SCODA based purification, described herein aselectrophoretic SCODA, time varying electric fields both provide aperiodic driving force and alter the drag (or equivalently the mobility)of molecules that have a mobility in the medium that depends on electricfield strength, e.g. nucleic acid molecules. For example, DNA moleculeshave a mobility that depends on the magnitude of an applied electricfield while migrating through a sieving matrix such as agarose orpolyacrylamide¹. By applying an appropriate periodic electric fieldpattern to a separation matrix (e.g. an agarose or polyacrylamide gel) aconvergent velocity field can be generated for all molecules in the gelwhose mobility depends on electric field. The field dependant mobilityis a result of the interaction between a reptating DNA molecule and thesieving matrix, and is a general feature of charged molecules with highconformational entropy and high charge to mass ratios moving throughsieving matrices. Since nucleic acids tend to be the only moleculespresent in most biological samples that have both a high conformationalentropy and a high charge to mass ratio, electrophoretic SCODA basedpurification has been shown to be highly selective for nucleic acids.

The ability to detect specific biomolecules in a sample has wideapplication in the field of diagnosing and treating disease. Researchcontinues to reveal a number of biomarkers that are associated withvarious disorders. Exemplary biomarkers include genetic mutations, thepresence or absence of a specific protein, the elevated or reducedexpression of a specific protein, elevated or reduced levels of aspecific RNA, the presence of modified biomolecules, and the like.Biomarkers and methods for detecting biomarkers are potentially usefulin the diagnosis, prognosis, and monitoring the treatment of variousdisorders, including cancer, disease, infection, organ failure and thelike.

The differential modification of biomolecules in vivo is an importantfeature of many biological processes, including development and diseaseprogression. One example of differential modification is DNAmethylation. DNA methylation involves the addition of a methyl group toa nucleic acid. For example a methyl group may be added at the 5′position on the pyrimidine ring in cytosine². Methylation of cytosine inCpG islands is commonly used in eukaryotes for long term regulation ofgene expression². Aberrant methylation patterns have been implicated inmany human diseases including cancer. DNA can also be methylated at the6 nitrogen of the adenine purine ring.

Chemical modification of molecules, for example by methylation,acetylation or other chemical alteration, may alter the binding affinityof a target molecule and an agent that binds the target molecule. Forexample, methylation of cytosine residues increases the binding energyof hybridization relative to unmethylated duplexes³⁻⁵. The effect issmall. Previous studies report an increase in duplex melting temperatureof around 0.7° C. per methylation site in a 16 nucleotide sequence⁴ whencomparing duplexes with both strands unmethylated to duplexes with bothstrands methylated.

There remains a need for methods and apparatus capable of providingimproved separation and purification of molecules, including identicalmolecules that are differentially modified.

SUMMARY

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods which aremeant to be exemplary and illustrative, not limiting in scope. Invarious embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

One embodiment provides a method for concentrating a molecule ofinterest from a biological sample. A biological sample is obtained fromthe subject and loaded on an affinity matrix. The affinity matrix has animmobilized affinity agent that has a first binding affinity for themolecule of interest and a second binding affinity for at least some ofthe other molecules in the biological sample. The first binding affinityis higher than the second binding affinity. Affinity SCODA is conductedto selectively concentrate the molecule of interest into a focus spot,wherein the concentration of the molecule of interest in the focus spotis increased relative to the concentration of the other molecules in thebiological sample. The molecules may be nucleic acids. The molecule ofinterest may have the same sequence as at least some of the othermolecules in the biological sample. The molecule of interest may bedifferentially modified as compared to at least some of the othermolecules in the biological sample. The molecule of interest may bedifferentially methylated as compared to at least some of the othermolecules in the biological sample. The biological sample may bematernal plasma and the molecule of interest may be fetal DNA that isdifferentially methylated as compared to maternal DNA. The biologicalsample may be a tissue sample and the molecule of interest may be a genethat is implicated in cancer that is differentially methylated ascompared to the gene in a healthy subject.

One embodiment provides a method for separating a first molecule from asecond molecule in a sample. An affinity matrix is provided withimmobilized probes that bind to the first and second molecules. Abinding energy between the first molecule and the probe is greater thana binding energy between the second molecule and the probe. A spatialgradient that is a mobility altering field that alters the affinity ofthe first and second molecules for the probe is provided within theaffinity matrix. A driving field that effects motion of the moleculeswithin the affinity matrix is applied. The orientation of both thespatial gradient and the driving field is varied over time to effect netmotion of the first molecule towards a focus spot. A washing field isapplied and is positioned to effect net motion of both the first andsecond molecules through the affinity matrix. The first and secondmolecules may be nucleic acids. The first and second molecules may bedifferentially modified. The first and second molecules may bedifferentially methylated. The first molecule may be fetal DNA and thesecond molecule may be maternal DNA that has the same sequence as thefetal DNA but is differentially methylated as compared to the fetal DNA.The first molecule and the second molecule may be a gene that isimplicated in cancer, and the first molecule may be differentiallymethylated as compared to the second molecule.

One embodiment provides the use of a time-varying driving field incombination with a time-varying mobility altering field to separatefirst and second differentially methylated nucleic acid molecules,wherein the first and second nucleic acid molecules have the same DNAsequence. A time-varying driving field and a time-varying mobilityaltering field are applied to a matrix including an oligonucleotideprobe that is at least partially complementary to said DNA sequence. Thefirst nucleic acid molecule has a first binding energy to theoligonucleotide probe and the second nucleic acid molecule has a secondbinding energy to the oligonucleotide probe, and the first bindingenergy is higher than the second binding energy. The first nucleic acidmolecules may be fetal DNA, the second nucleic acid molecules may bematernal DNA, and the first and second nucleic acid molecules may beobtained from a sample of maternal blood. The first and second nucleicacid molecules may be a gene that is implicated in a fetal disorder. Thefirst and second molecules may be differentially methylated forms of agene that is implicated in cancer. The first and second molecules may beobtained from a tissue sample of a subject.

One embodiment provides the use of synchronous coefficient of dragalteration (SCODA) to detect the presence of a biomarker in a subject.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following detailed descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures of thedrawings. The embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 shows a plot of equation [10] showing the SCODA drift velocity inone dimension over the domain extending from −L to +L.

FIG. 2 shows a plot of equation [23] near the duplex melting temperatureT_(m) illustrating the relative change in mobility as a function oftemperature.

FIG. 3 shows a plot of mobility versus temperature for two differentmolecules with different binding energies to immobilized probemolecules. The mobility of the high binding energy target is shown bythe curve on the right, while the mobility of the low binding energytarget is shown by the curve on the left.

FIG. 4 shows the effect of an applied DC washing bias on molecules withtwo different binding energies. The solid curve represents the driftvelocity of a target molecule with a lower binding energy to the boundprobes than the molecules represented by the dashed curve.

FIG. 5 shows an example of an electric field pattern suitable for twodimensional SCODA based concentration in some embodiments. Voltagesapplied at electrodes A, B, C and D, are −V, 0, 0, and 0 respectively.Arrows represent the velocity of a negatively charged analyte moleculesuch as DNA. Colour intensity represents electric field strength.

FIG. 6 shows stepwise rotation of the electric field leading to focusingof molecules whose mobility increases with temperature in one embodimentof affinity SCODA. A particle path is shown by the arrows.

FIG. 7 shows the gel geometry including boundary conditions and bulk gelproperties used for electrothermal modeling.

FIG. 8 shows the results of an electrothermal model for a single step ofthe SCODA cycle in one embodiment. Voltage applied to the fourelectrodes was −120 V, 0 V, 0 V, 0 V. Spreader plate temperature was setto 55° C. (328 K).

FIG. 9 shows SCODA velocity vector plots in one exemplary embodiment ofthe invention.

FIGS. 10A and 10B show predictions of SCODA focusing under theapplication of a DC washing bias in one embodiment. FIG. 10A shows theSCODA velocity field for perfect match target. A circular spot indicatesfinal focus location. FIG. 10B shows the SCODA velocity field for thesingle base mismatch target.

FIG. 11 shows the results of the measurement of temperature dependenceof DNA target mobility through a gel containing immobilizedcomplementary oligonucleotide probes for one exemplary separation.

FIG. 12 shows a time series of affinity SCODA focusing under theapplication of DC bias according to one embodiment. Perfect match DNA istagged with 6-FAM (green) (leading bright line that focuses to a tightspot) and single base mismatch DNA is tagged with Cy5 (red) (trailingbright line that is washed from the gel). Images taken at 3 minuteintervals. The first image was taken immediately following injection.

FIGS. 13A, 13B, 13C and 13D show the results of performing SCODAfocusing with different concentrations of probes and in the presence orabsence of 200 mM NaCl. Probe concentrations are 100 μM, 10 μM, 1 μM,and 100 μM, respectively. The buffer used in FIGS. 13A, 13B and 13C was1×TB with 0.2 M NaCl. The buffer used in FIG. 13D was 1×TBE. Differentamounts of target were injected in each of these experiments, and thecamera gain was adjusted prevent saturation.

FIG. 14 shows an experiment providing an example of phase lag inducedrotations. The field rotation is counterclockwise, that induces aclockwise rotation of the targets in the gel. Images were taken at 5minute intervals.

FIG. 15A shows the focus location under bias for 250 bp and 1000 bpfragments labeled with different fluorescent markers, with squaresindicating data for the application of a 10 V DC bias and circlesindicating data for the application of a 20 V DC bias. FIG. 15B shows animage of the affinity gel at the end of the run, wherein images showingthe location of each fluorescent marker have been superimposed.

FIGS. 16A and 16B show respectively the normalized fluorescence signaland the calculated rejection ratio of a 100 nucleotide sequence having asingle base mismatch as compared with a target DNA molecule according toone example.

FIGS. 17A, 17B and 17C show enrichment of cDNA obtained from an EZH2Y641N mutation from a mixture of wild type and mutant amplicons usingaffinity SCODA with the application of a DC bias. Images were taken at 0minutes (FIG. 17A), 10 minutes (FIG. 17B), and 20 minutes (FIG. 17C).

FIG. 18 shows experimental results for the measurement of mobilityversus temperature for methylated and unmethylated targets. Data pointswere fit to equation [23]. Data for the unmethylated target is fit tothe curve on the left; data for the methylated target is fit to thecurve on the right.

FIG. 19 shows the difference between the two mobility versus temperaturecurves which were fit to the data from FIG. 18. The maximum value ofthis difference is at 69.5° C., which is the temperature for maximumseparation while performing affinity SCODA focusing with the applicationof a DC bias.

FIG. 20 shows experimental results for the separation of methylated(6-FAM, green) and unmethylated (Cy5, red) targets by using SCODAfocusing with an applied DC bias.

FIGS. 21A-21D show the separation of differentially methylatedoligonucloetides using affinity SCODA. FIGS. 21A and 21B show theresults of an initial focus before washing unmethylated target from thegel for 10 pmol unmethylated DNA (FIG. 21A) and 0.1 pmol methylated DNA(FIG. 21B). FIGS. 21C and 21D show the results of a second focusingconducted after the unmethylated sequence had been washed from the gelfor unmethylated and methylated target, respectively.

FIGS. 22A-22K show the results of the differential separation of twodifferent sequences in the same affinity matrix using differentoligonucleotide probes. FIG. 22A shows the gel after loading. FIGS. 22Band 22C show focusing at 55° C. after 2 minutes and 4 minutes,respectively. FIGS. 22D and 22E show focusing at 62° C. after 2 minutesand 4 minutes, respectively. FIGS. 22 F, 22G and 22H show focusing ofthe target molecules to an extraction well at the centre of the gelafter 0.5 minutes and 1 minute at 55° C. and at 3 minutes after raisingthe temperature to 62° C., respectively. FIGS. 22I, 22J and 22K show theapplication of a washing bias to the right at 55° C. after 6 minutes, 12minutes and 18 minutes, respectively.

DETAILED DESCRIPTION

Throughout the following description specific details are set forth inorder to provide a more thorough understanding to persons skilled in theart. However, well known elements may not have been shown or describedin detail to avoid unnecessarily obscuring the disclosure. Accordingly,the description and drawings are to be regarded in an illustrative,rather than a restrictive, sense.

As used herein, the term “differentially modified” means two moleculesof the same kind that have been chemically modified in different ways.Non-limiting examples of differentially modified molecules include: aprotein or a nucleic acid that has been methylated is differentiallymodified as compared with the unmethylated molecule; a nucleic acid thatis hypermethylated or hypomethylated (e.g. as may occur in cancerous orprecancerous cells) is differentially modified as compared with thenucleic acid in a healthy cell; a histone that is acetylated isdifferentially modified as compared with the non-acetylated histone; andthe like.

In some embodiments, molecules that are differentially modified areidentical to one another except for the presence of a chemicalmodification on one of the molecules. In some embodiments, moleculesthat are differentially modified are very similar to one another, butnot identical. For example, where the molecules are nucleic acids orproteins, one of the biomolecules may share at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% sequence identity with thedifferentially modified molecule.

Affinity SCODA

SCODAphoresis is a previously documented method for injectingbiomolecules into a gel, and preferentially concentrating nucleic acidsor other biomolecules of interest in the center of the gel. SCODA may beapplied, for example, to DNA, RNA and other molecules. Followingconcentration, the purified molecules may be removed for furtheranalysis. In one specific embodiment of SCODAphoresis—affinitySCODA—binding sites which are specific to the biomolecules of interestmay be immobilized in the gel. In doing so one may be able generate anon-linear motive response to an electric field for biomolecules thatbind to the specific binding sites. One specific application of affinitySCODA is sequence-specific SCODA. Here oligonucleotides may beimmobilized in the gel allowing for the concentration of only DNAmolecules which are complementary to the bound oligonucleotides. Allother DNA molecules which are not complementary may focus weakly or notat all and can therefore be washed off the gel by the application of asmall DC bias.

SCODA based transport is a general technique for moving particlesthrough a medium by first applying a time-varying forcing (i.e. driving)field to induce periodic motion of the particles and superimposing onthis forcing field a time-varying perturbing field that periodicallyalters the drag (or equivalently the mobility) of the particles (i.e. amobility-altering field). Application of the mobility-altering field iscoordinated with application of the forcing field such that theparticles will move further during one part of the forcing cycle than inother parts of the forcing cycle. Specifically, the drift velocity υ(t)of a particle driven by an external force F(t) with a time varying dragcoefficient ζ(t) (i.e. a varying mobility) is given by:

$\begin{matrix}{{v(t)} = \frac{F(t)}{\zeta (t)}} & \lbrack 1\rbrack\end{matrix}$

If the external force and drag coefficient vary periodically such that

$\begin{matrix}{{{F(t)} = {F_{0}\mspace{14mu} {\sin \left( {\omega \; t} \right)}}}{{and},}} & \lbrack 2\rbrack \\{\frac{1}{\zeta (t)} = {\frac{1}{\zeta_{0}} + \frac{\sin \left( {{\omega \; t} + \varphi} \right)}{\zeta_{1}}}} & \lbrack 3\rbrack\end{matrix}$

then the drift velocity averaged over one complete cycle is given by:

$\begin{matrix}{{\overset{\_}{v}(t)} = {\frac{F_{0}}{2\zeta_{1}}{\cos (\varphi)}}} & \lbrack 4\rbrack\end{matrix}$

By varying the drag (i.e. mobility) of the particle at the samefrequency as the external applied force, a net drift can be induced withzero time-averaged forcing. The result of equation [4] can be used withan appropriate choice of driving force and drag coefficients that varyin time and space to generate a convergent velocity field in one or twodimensions. A time varying drag coefficient and driving force can beutilized in a real system to specifically concentrate (i.e.preferentially focus) only certain molecules, even where the differencesbetween the target molecule and one or more non-target molecules arevery small, e.g. molecules that are differentially modified at one ormore locations, or nucleic acids differing in sequence at one or morebases.

One Dimensional SCODA Concentration

By combining a spatially uniform driving force that varies periodicallyin time, with a drag coefficient that varies in time as well as in spaceit is possible to generate a convergent velocity field in one dimension.Consider the case of a charged particle with mobility μ moving under theinfluence of an applied electric field E; its velocity will be given by:

υ(x,t)=μ(x,t)E(x,t)  [5]

If electric field is varied periodically in time such that:

E(x,t)=E ₀ sin(ωt)  [6]

and a linear mobility gradient is provided within the domain −L≦x≦L thatvaries at the same period:

μ(x,t)=μ₀+(kx)sin(ωt+φ)  [7]

where k can be thought of as the amplitude of the mobility variation,SCODA-based separation of particles can be achieved.

There are a number of ways to establish a mobility gradient for chargedmolecules moving in solution under the influence of an applied externalelectric field. For example, a time-varying electric field may beprovided as described above, a temperature gradient may be established,a pH gradient may be established, a light gradient may be establishedfor molecules which undergo a conformational change in the presence orabsence of light, or the like.

With the mobility gradient of equation [7] provided, the velocitybecomes:

υ(x,t)=[μ₀(kx)sin(ωt+φ)][E ₀ sin(ωt)]  [8]

Taking the time average of this velocity over one complete cycle yieldsthe following drift velocity:

$\begin{matrix}{{{\overset{\_}{v}}_{d}\left( {x,t} \right)} = {\frac{\omega}{2\pi}{\int_{0}^{\frac{2\pi}{\omega}}{{v\left( {x,t} \right)}\ {t}}}}} & \lbrack 9\rbrack \\{{{\overset{\_}{v}}_{d}\left( {x,t} \right)} = {\frac{kx}{2}E_{0}\mspace{14mu} {\cos (\varphi)}}} & \lbrack 10\rbrack\end{matrix}$

This velocity field has an equilibrium point at x=0 and can be madeconvergent or divergent depending on the sign of kE₀ cos(φ). Forpositive values the velocity field is divergent and for negative valuesit is convergent. FIG. 1 shows the velocity plotted as a function of xfor the case where kE₀ cos(φ)<0. The arrows in FIG. 1 indicate thedirection of drift. All particles between −L and +L will drift towardsthe zero velocity point at x=0. Outside of the domain the time averagedvelocity is zero as the mobility is only altered between −L and +L.

In the embodiment illustrated in FIG. 1, the velocity takes on apositive value for negative values of x and vice versa for positivevalues of x resulting in all particles within the domain driftingtowards x=0 where the velocity is zero.

Two Dimensional SCODA

To extend the result of equation [10] to two dimensions, in someembodiments a rotating electric field is used as the driving field and arotating mobility gradient is established:

{right arrow over (E)}=E ₀ cos(ωt)î−E ₀ sin(ωt)ĵ  [11]

μ=μ₀ +k[x cos(ωt+φ)−y sin(ωt+φ)]  [12]

As in the one dimensional case {right arrow over (υ)}=μ{right arrow over(E)}, and the same integration as in equation [9] can be performed toyield the time averaged drift velocity in two dimensions:

$\begin{matrix}{{\overset{\_}{v}}_{x} = {\frac{\omega}{2\pi}{\int_{0}^{\frac{2\pi}{\omega}}{E_{0}\mspace{14mu} {\cos \left( {\omega \; t} \right)}\left( {\mu_{0} + {k\left( {{x\mspace{14mu} {\cos \left( {{\omega \; t} + \varphi} \right)}} - {y\mspace{14mu} {\sin \left( {{\omega \; t} + \varphi} \right)}}} \right)}} \right)\ {t}}}}} & \lbrack 13\rbrack \\{{\overset{\_}{v}}_{y} = {\frac{\omega}{2\pi}{\int_{0}^{\frac{2\pi}{\omega}}{{- E_{0}}\mspace{14mu} {\cos \left( {\omega \; t} \right)}\left( {\mu_{0} + {k\left( {{x\mspace{14mu} {\cos \left( {{\omega \; t} + \varphi} \right)}} - {y\mspace{14mu} {\sin \left( {{\omega \; t} + \varphi} \right)}}} \right)}} \right)\ {t}}}}} & \lbrack 14\rbrack\end{matrix}$

This results in the following expression for the drift velocity:

$\begin{matrix}{\overset{\rightharpoonup}{v} = {\frac{E_{0}k}{2}\left( {{\left( {{x\mspace{14mu} {\cos (\varphi)}} - {y\mspace{14mu} {\sin (\varphi)}}} \right)\hat{i}} + {\left( {{x\mspace{14mu} {\sin (\varphi)}} + {y\mspace{14mu} {\cos (\varphi)}}} \right)\hat{j}}} \right)}} & \lbrack 15\rbrack\end{matrix}$

Rewriting in polar coordinates and simplifying yields:

$\begin{matrix}{\overset{\rightharpoonup}{v} = {\frac{E_{0}{kr}}{2}\left( {{{\cos (\varphi)}\hat{r}} + {{\sin (\varphi)}\hat{\theta}}} \right)}} & \lbrack 16\rbrack\end{matrix}$

This result highlights a number of aspects of SCODA in two dimensions.It shows that despite the zero time averaged forcing there will benon-zero drift everywhere except at the point in the medium where r=0.It shows that the nature of the drift depends on the relative phase, φ,of the two signals, with the strength of focusing (the radial,{circumflex over (r)}, term) being proportional to the cosine of thephase lag between the electric driving field oscillations and themobility oscillations. For a 0° phase angle there is a purely focusingvelocity field with net drift directed towards the centre of the domain.For a 180° phase angle the velocity field is pure de-focusing with netdrift away from the centre of the gel. And for phase angles of 90° and270° the velocity field is purely rotational. At intermediate angles theresultant velocity field will be a combination of both rotational andfocusing components. To achieve efficient focusing, in some embodimentsthe phase difference between the driving force and the mobilityvariation is as small as possible.

Generation of a Time Varying Mobility Field

Previous applications of SCODA based concentration used the fact thatthe mobility of DNA in a sieving matrix such as agarose orpolyacrylamide depends on the magnitude of the applied electric field.In some applications, the molecules of interest may have a mobility thatdoes not normally depend strongly on electric field, such as shortnucleic acids less than 200 bases, biomolecules other than nucleic acids(e.g. proteins or polypeptides), or the like. In some applications, itmay be desired to purify only a subset of the nucleic acids in a sample,for example purifying or detecting a single gene from a sample ofgenomic DNA or purifying or detecting a chemically modified molecule(e.g. methylated DNA) from a differentially modified molecule having thesame basic structure (e.g. unmethylated DNA having the same sequence),or the like.

SCODA-based purification of molecules that do not have a mobility thatis strongly dependent on electrical field strength (i.e. which have alow value of k based on variations in electric field strength) can beachieved by using a SCODA matrix that has an affinity to the molecule tobe concentrated. An affinity matrix can be generated by immobilizing anagent with a binding affinity to the target molecule (i.e. a probe) in amedium. Using such a matrix, operating conditions can be selected wherethe target molecules transiently bind to the affinity matrix with theeffect of reducing the overall mobility of the target molecule as itmigrates through the affinity matrix. The strength of these transientinteractions is varied over time, which has the effect of altering themobility of the target molecule of interest. SCODA drift can thereforebe generated. This technique is called affinity SCODA, and is generallyapplicable to any target molecule that has an affinity to a matrix.

Affinity SCODA can selectively enrich for nucleic acids based onsequence content, with single nucleotide resolution. In addition,affinity SCODA can lead to different values of k for molecules withidentical DNA sequences but subtly different chemical modifications suchas methylation. Affinity SCODA can therefore be used to enrich for (i.e.preferentially focus) molecules that differ subtly in binding energy toa given probe, and specifically can be used to enrich for methylated,unmethylated, hypermethylated, or hypomethylated sequences.

Exemplary media that can be used to carry out affinity SCODA include anymedium through which the molecules of interest can move, and in which anaffinity agent can be immobilized to provide an affinity matrix. In someembodiments, polymeric gels including polyacrylamide gels, agarose gels,and the like are used. In some embodiments, microfabricated/microfluidicmatrices are used.

Exemplary operating conditions that can be varied to provide a mobilityaltering field include temperature, pH, salinity, concentration ofdenaturants, concentration of catalysts, application of an electricfield to physically pull duplexes apart, or the like.

Exemplary affinity agents that can be immobilized on the matrix toprovide an affinity matrix include nucleic acids having a sequencecomplementary to a nucleic acid sequence of interest, proteins havingdifferent binding affinities for differentially modified molecules,antibodies specific for modified or unmodified molecules, nucleic acidaptamers specific for modified or unmodified molecules, other moleculesor chemical agents that preferentially bind to modified or unmodifiedmolecules, or the like.

The affinity agent may be immobilized within the medium in any suitablemanner. For example where the affinity agent is an oligonucleotide, theoligonucleotide may be covalently bound to the medium, acrydite modifiedoligonucleotides may be incorporated directly into a polyacrylamide gel,the oligonucleotide may be covalently bound to a bead or other constructthat is physically entrained within the medium, or the like.

Where the affinity agent is a protein or antibody, in some embodimentsthe protein may be physically entrained within the medium (e.g. theprotein may be cast directly into an agarose or polyacrylamide gel),covalently coupled to the medium (e.g. through use of cyanogen bromideto couple the protein to an agarose gel), covalently coupled to a beadthat is entrained within the medium, bound to a second affinity agentthat is directly coupled to the medium or to beads entrained within themedium (e.g. a hexahistidine tag bound to NTA-agarose), or the like.

Where the affinity agent is a protein, the conditions under which theaffinity matrix is prepared and the conditions under which the sample isloaded should be controlled so as not to denature the protein (e.g. thetemperature should be maintained below a level that would be likely todenature the protein, and the concentration of any denaturing agents inthe sample or in the buffer used to prepare the medium or conduct SCODAfocusing should be maintained below a level that would be likely todenature the protein).

Where the affinity agent is a small molecule that interacts with themolecule of interest, the affinity agent may be covalently coupled tothe medium in any suitable manner.

One exemplary embodiment of affinity SCODA is sequence-specific SCODA.In sequence specific SCODA, the target molecule is or comprises anucleic acid molecule having a specific sequence, and the affinitymatrix contains immobilized oligonucleotide probes that arecomplementary to the target nucleic acid molecule. In some embodiments,sequence specific SCODA is used both to separate a specific nucleic acidsequence from a sample, and to separate and/or detect whether thatspecific nucleic acid sequence is differentially modified within thesample. In some such embodiments, affinity SCODA is conducted underconditions such that both the nucleic acid sequence and thedifferentially modified nucleic acid sequence are concentrated by theapplication of SCODA fields. Contaminating molecules, including nucleicacids having undesired sequences, can be washed out of the affinitymatrix during SCODA focusing. A washing bias can then be applied inconjunction with SCODA focusing fields to separate the differentiallymodified nucleic acid molecules as described below by preferentiallyfocusing the molecule with a higher binding energy to the immobilizedoligonucleotide probe.

Mobility of a Target in an Affinity Matrix

The interactions between a target and immobilized probes in an affinitymatrix can be described by first order reaction kinetics:

$\begin{matrix}{{\lbrack T\rbrack + \lbrack P\rbrack}\underset{k_{r}}{\overset{k_{f}}{\rightleftharpoons}}\left\lbrack {T\mspace{14mu} \ldots \mspace{14mu} P} \right\rbrack} & \lbrack 17\rbrack\end{matrix}$

Here [T] is the target, [P] the immobilized probe, [T. P] theprobe-target duplex, k_(f) is the forward (hybridization) reaction rate,and k_(r) the reverse (dissociation) reaction rate. Since the mobilityof the target is zero while it is bound to the matrix, the effectivemobility of the target will be reduced by the relative amount of targetthat is immobilized on the matrix:

$\begin{matrix}{\mu_{effective} = {\mu_{0}\frac{\lbrack T\rbrack}{\lbrack T\rbrack + \left\lbrack {T\mspace{14mu} \ldots \mspace{14mu} P} \right\rbrack}}} & \lbrack 18\rbrack\end{matrix}$

where μ₀ is the mobility of the unbound target. Using reasonableestimates for the forward reaction rate⁶ and an immobilized probeconcentration that is significantly higher than the concentration of theunbound target, it can be assumed that the time constant forhybridization should be significantly less than one second. If theperiod of the mobility-altering field is maintained at longer than onesecond, it can be assumed for the purposes of analysis that the bindingkinetics are fast and equation [17] can be rewritten in terms ofreaction rates:

$\begin{matrix}{{{k_{f}\lbrack T\rbrack}\lbrack P\rbrack} = {k_{r}\left\lbrack {T\mspace{14mu} \ldots \mspace{14mu} P} \right\rbrack}} & \lbrack 19\rbrack \\{\lbrack T\rbrack = {\frac{k_{r}}{k_{f}}\frac{\left\lbrack {T\mspace{14mu} \ldots \mspace{14mu} P} \right\rbrack}{\lbrack P\rbrack}}} & \lbrack 20\rbrack\end{matrix}$

Inserting [20] into equation [18] and simplifying yields:

$\begin{matrix}{\mu_{effective} = {\mu_{0}\frac{1}{1 + {\frac{k_{f}}{k_{r}}\lbrack P\rbrack}}}} & \lbrack 21\rbrack\end{matrix}$

From this result it can be seen that the mobility can be altered bymodifying either the forward or reverse reaction rates. Modification ofthe forward or reverse reaction rates can be achieved in a number ofdifferent ways, for example by adjusting the temperature, salinity, pH,concentration of denaturants, concentration of catalysts, by physicallypulling duplexes apart with an external electric field, or the like. Inone exemplary embodiment described in greater detail below, themechanism for modifying the mobility of target molecules moving throughan affinity matrix is control of the matrix temperature.

To facilitate analysis, it is helpful to make some simplifyingassumptions. First it is assumed that there are a large number ofimmobilized probes relative to target molecules. So long as this istrue, then even if a large fraction of the target molecules become boundto the probes the concentration of free probes, [P], will not changemuch and it can be assumed that [P] is constant. Also, it is assumedthat the forward reaction rate k_(f) does not depend on temperature.This not strictly true, as the forward reaction rate does depend ontemperature^(7,8). Secondary structure in the immobilized probe or inthe target molecule can result in a temperature dependant forwardreaction rate⁹. However, in embodiments operating at a temperature rangenear the duplex melting temperature the reverse reaction rate has anexponential dependence on temperature and the forward reaction rate hasa much weaker temperature dependence, varying by about 30% over a rangeof 30° C. around the melting temperature¹⁰. It is additionally assumedthat the target sequence is free of any significant secondary structure.Although this final assumption would not always be correct, itsimplifies this initial analysis.

To determine the temperature dependence of the reverse reaction rate, anArrhenius model for unbinding kinetics is assumed. This assumption isjustified by recent work in nanopore force spectroscopy^(11,12).

$\begin{matrix}{k_{r} = {A\; ^{\frac{\Delta \; G}{k_{b}T}}}} & \lbrack 22\rbrack\end{matrix}$

Here A is an empirically derived constant, ΔG is the probe-targetbinding energy, k_(b) is the Boltzmann constant, and T the temperature.Inserting this into [21], rewriting the free energy ΔG as ΔH−TΔS, andcollecting constant terms allows the mobility to be rewritten as:

$\begin{matrix}{\mu_{effective} = {\mu_{0}\frac{1}{1 + {\beta }^{\frac{{{- \Delta}\; H} + {T\; \Delta \; S}}{k_{b}T}}}}} & \lbrack 23\rbrack\end{matrix}$

Equation [23] describes a sigmoidal mobility temperature dependence. Theshape of this curve is shown in FIG. 2. At low temperature the mobilityis nearly zero. This is the regime where thermal excitations areinsufficient to drive target molecules off of the affinity matrix. Athigh temperature target molecules move at the unbound mobility, wherethe thermal energy is greater than the binding energy. Between these twoextremes there exists a temperature range within which a small change intemperature results in a large change in mobility. This is the operatingregime for embodiments of affinity SCODA that utilize temperature as themobility altering parameter.

In embodiments of affinity SCODA used to separate nucleic acids based onsequence, i.e. sequence-specific SCODA, this temperature range tends tolie near the melting temperature of the probe-target duplex. Equations[10] and [16] state that the speed of concentration is proportional tok, which is a measure of how much the mobility changes during one SCODAcycle. Operating near the probe-target duplex melting temperature, wherethe slope of the mobility versus temperature curve is steepest,maximizes k for a given temperature swing during a SCODA cycle inembodiments where temperature is used as the mobility alteringparameter.

In some embodiments, affinity SCODA may be conducted within atemperature gradient that has a maximum amplitude during application ofSCODA focusing fields that varies within about ±20° C., within about±10° C., within about ±5° C., or within about ±2° C. of the meltingtemperature of the target molecule and the affinity agent.

It is possible to describe affinity SCODA in one dimension by replacingthe time dependent mobility of equation [7] with the temperaturedependent mobility of equation [23] and a time dependent temperature:

$\begin{matrix}{{T\left( {x,t} \right)} = {T_{m} + {{T_{a}\left( \frac{x}{L} \right)}{\sin \left( {{\omega \; t} + \varphi} \right)}}}} & \lbrack 24\rbrack\end{matrix}$

Here, the temperature oscillates around T_(m), the probe target meltingtemperature, and T_(a) is the maximum amplitude of the temperatureoscillations at x=±L. To get an analytical expression for the driftvelocity, υ_(d)=μE, as a function of temperature, a Taylor expansion ofequation [23] is performed around T_(m):

$\begin{matrix}{\mu_{effective} = {{\mu \left( T_{m} \right)} - {\frac{\mu_{0}\beta \; \Delta \; H\; ^{\frac{{{- \Delta}\; H} + {T\; {\Delta S}}}{k_{b}T_{m}}}}{k_{b}{T_{m}^{2}\left( {1 + {\beta }^{\frac{{{- \Delta}\; H} + {T\; \Delta \; S}}{k_{b}T_{m}}}} \right)}^{2}}\left( {T - T_{m}} \right)} + {O\left( \left( {T - T_{m}} \right)^{2} \right)}}} & \lbrack 25\rbrack\end{matrix}$

which can be rewritten as:

μeffective=μ(T _(m))+α(T−T _(m))+O((T−T _(m))²)  [26]

Here the first term in the Taylor expansion has been collected into theconstant α. Combining [24] and [26] into an expression for the mobilityyields an expression similar to [7]:

$\begin{matrix}{{\mu (t)} = {{\mu \left( T_{m} \right)} + {\left( \frac{\alpha \; T_{a}x}{L} \right){\sin \left( {{\omega \; t} + \varphi} \right)}}}} & \lbrack 27\rbrack\end{matrix}$

Equation [27] can be used to determine the time averaged drift velocityfor both the one dimensional and two dimensional cases by simplyreplacing k with:

$\begin{matrix}{{\alpha \; \frac{T_{a}}{L}} = {\frac{\mu_{0}\beta \; \Delta \; H\; ^{\frac{{{- \Delta}\; H} + {T\; \Delta \; S}}{k_{b}T_{m}}}}{k_{b}{T_{m}^{2}\left( {1 + {\beta }^{\frac{{{- \Delta}\; H} + {T\; \Delta \; S}}{k_{b}T_{m}}}} \right)}^{2}}\left( \frac{T_{a}}{L} \right)}} & \lbrack 28\rbrack\end{matrix}$

The drift velocity is then given by:

$\begin{matrix}{{{\overset{\_}{v}}_{d}\left( {x,t} \right)} = {\frac{\alpha \; T_{a}x}{2\; L}E_{0}{\cos (\varphi)}}} & \lbrack 29\rbrack\end{matrix}$

in one dimension, and:

$\begin{matrix}{\overset{->}{v} = {\frac{E_{0}\alpha \; T_{a}r}{2L}\left( {{{\cos (\varphi)}\hat{r}} + {{\sin (\varphi)}\hat{\theta}}} \right)}} & \lbrack 30\rbrack\end{matrix}$

in two dimensions. This result shows that if a two dimensional gelfunctionalized with immobilized probes (i.e. an affinity matrix), thenby combining a rotating temperature gradient with a rotating dipoleelectric field, all target molecules should be forced towards a centralregion in the gel, thus concentrating a target molecule that binds tothe immobilized probes.Molecular Separation with Affinity SCODA

In some embodiments, affinity SCODA is used to separate two similarmolecules (e.g. the same molecule that has been differentially modified,or which differs in sequence at only one or a few locations) withdiffering binding affinities for the immobilized probe. Beginning withtwo molecular species, each with a different binding energy to theimmobilized probes, these two molecular species can be separated bysuperimposing a washing motive force over the driving and mobilityaltering fields used to produce SCODA focusing, to provide net motion ofmolecules that have a lesser binding affinity for the immobilized probe(i.e. the molecules that have a higher binding affinity for theimmobilized probe are preferentially focused during the application ofthe SCODA focusing fields). In some embodiments, the washing force is asmall applied DC force, referred to herein as a DC bias.

In the one dimensional case when a small DC force is applied as awashing or bias force, the electric field becomes:

E(x,t)=E ₀ sin(ωt)+E _(b)  [31]

where E_(b) is the applied DC bias. The final drift velocity hassuperimposed on the SCODA focusing velocity a constant velocityproportional to the strength of the bias field:

$\begin{matrix}{{{\overset{\_}{v}}_{d}\left( {x,t} \right)} = {{\frac{\alpha \; T_{a}x}{2L}E_{0}{\cos (\varphi)}} + {{\mu \left( T_{m} \right)}E_{b}}}} & \lbrack 32\rbrack\end{matrix}$

This drift velocity will tend to move the final focus location either tothe left or right depending on the direction of bias. The amount bywhich this bias moves a focus off centre depends on the strength of theinteraction between the target and probe molecules. The differentialstrength of the target-probe interaction can therefore serve as amechanism to enable molecular separation of two highly similar species.

Consider two molecules that have different binding affinities for animmobilized probe. Reducing the probe-target binding energy, ΔG inequation [23], will serve to shift the mobility versus temperature curveto the left on the temperature scale as shown in FIG. 3. The mobility ofthe high binding energy target is shown by the curve on the right, whilethe mobility of the low binding energy target is shown by the curve onthe left.

If the SCODA system in this exemplary embodiment is operated at theoptimal focusing temperature for the higher binding energy molecule,T_(m) in FIG. 3, then the mobility of the lower binding energy moleculewill be higher and will have weaker temperature dependence. In terms ofequation [32] the molecule with lower binding energy will have a largervalue of μ(T_(m)) and a smaller value of a. This means that a lowerbinding energy molecule will have a lower SCODA drift velocity and ahigher velocity under DC bias, resulting in a different final focuslocation than the high binding energy molecule as illustrated in FIG. 4.

FIG. 4 shows the effect of an applied DC bias on molecules with twodifferent binding energies for the immobilized probe according to oneembodiment. The solid curve represents the drift velocity of a targetmolecule with a lower binding energy to the bound probes than themolecules represented by the dashed curve. The final focus location isthe point where the drift velocity is equal to zero. The moleculesrepresented by the solid curve have both a lower SCODA drift velocityand a higher DC velocity compared to the molecules represented by thedashed curve. When SCODA focusing is combined with a DC bias the lowerbinding energy molecules will focus further away from the unbiased focusat x=0, resulting in two separate foci, one for each molecular species.The final focus position for the high binding energy molecule isindicated by reference numeral 30. The final focus position for the lowbinding energy molecule is indicated by reference numeral 32.

The two dimensional case is the same as the one dimensional case, thesuperimposed velocity from the applied washing bias moves the finalfocus spot off centre in the direction of the washing bias.

In some embodiments, if the difference in binding energies between themolecules to be separated is large enough and a sufficiently highwashing bias is applied, the low binding energy molecules can be washedoff of the affinity matrix while molecules with higher binding energyare retained in the affinity matrix, and may be captured at a focuslocation within the affinity matrix (i.e. preferentially focused)through the application of SCODA focusing fields.

Generation of a Time Varying Temperature Gradient

Embodiments of affinity SCODA that use variations in temperature as themobility altering field may use a periodically varying temperaturegradient to produce a convergent velocity field. A periodically varyingtemperature gradient may be provided in any suitable manner, for exampleby the use of heaters or thermoelectric chillers to periodically heatand cool regions of the medium, the use of radiative heating toperiodically heat regions of the medium, the application of light orradiation to periodically heat regions of the medium, Joule heatingusing the application of an electric field to the medium, or the like.

A periodically varying temperature gradient can be established in anysuitable manner so that particles that are spaced a farther distancefrom a desired focus spot experience greater mobility (i.e. are at ahigher temperature and hence travel farther) during times of applicationof the driving field towards the desired focus spot than during times ofapplication of the driving field away from the desired focus spot. Insome embodiments, the temperature gradient is rotated to produce aconvergent velocity field in conjunction with the application of atime-varying driving force.

In some embodiments, Joule heating using an electric field is used toprovide a temperature gradient. In some embodiments, the electric fieldused to provide Joule heating to provide a temperature gradient is thesame as the electric field that provides the driving field. In someembodiments, the magnitude of the electric field applied is selected toproduce a desired temperature gradient within an affinity matrix.

In some embodiments, a spatial temperature gradient is generated using aquadrupole electric field to provide the Joule heating. In some suchembodiments, a two dimensional gel with four electrodes is provided.Voltages are applied to the four electrodes such that the electric fieldin the gel is non-uniform, containing regions of high electric field(and consequently high temperature) and low electric field. The electricfield is oriented such that the regions of high electric field tend topush negatively charged molecules towards the centre of the gel, whileregions of low electric field tend to push such molecules away from thecentre of the gel. In some such embodiments, the electric field thatprovides the temperature gradient through Joule heating is also theelectric field that applies a driving force to molecules in the gel.

An example of such a field pattern is illustrated in FIG. 5. Voltagesapplied at electrodes A, B, C and D in FIG. 5 are −V, 0, 0, and 0respectively. Arrows represent the velocity of a negatively chargedanalyte molecule. Colour intensity represents electric field strength.The regions near electrode A have a high electric field strength, whichdecreases towards electrode C. The high field regions near electrode Atend to push negatively charged molecules towards the centre of the gel,while the lower field regions near electrodes B, C, and D tend to pushnegatively charged molecules away from the centre of the gel. Inembodiments in which the electric field also provides the temperaturegradient, the affinity matrix will become hotter in regions of higherfield strength due to Joule heating. Hence, regions of high electricfield strength will coincide with regions of higher temperature and thushigher mobility. Accordingly, molecules in the high electric fieldregions near electrode A will tend to move a greater distance toward thecentre of the gel, while molecules in the lower electric field regionsnear electrodes B, C, and D have a lower mobility (are at a coolertemperature) and will move only a short distance away from the centre ofthe gel.

In some embodiments, the electric field pattern of FIG. 5 is rotated ina stepwise manner by rotating the voltage pattern around the fourelectrodes such that the time averaged electric field is zero as shownin FIG. 6. This rotating field will result in net migration towards thecentre of the gel for any molecule that is negatively charged and has amobility that varies with temperature. In some embodiments, the electricfield pattern is varied in a manner other than rotation, e.g. bysequentially shifting the voltage pattern by 180°, 90°, 180°, and 90°,or by randomly switching the direction of the electric field. As shownabove, the mobility of a molecule moving through an affinity matrixdepends on temperature, not electric field strength. The appliedelectric field will tend to increase the temperature of the matrixthrough Joule heating; the magnitude of the temperature rise at anygiven point in the matrix will be proportional to the square of themagnitude of the electric field.

In embodiments in which the thermal gradient is provided by Jouleheating produced by the electric field that also provides the drivingfield, the oscillations in the thermal gradient will have the sameperiod as the electric field oscillations. These oscillations can driveaffinity SCODA based concentration in a two dimensional gel.

FIG. 6 illustrates the stepwise rotation of the electric field leadingto focusing of molecules whose mobility increases with temperature orelectric field according to such an embodiment. A particle path for anegatively charged molecule is shown. After four steps the particle hasa net displacement toward the centre of the gel. Molecules that do notexperience a change in mobility with changing temperature or electricfield will experience zero net motion in a zero time averaged electricfield.

Theoretical Predictions of Focusing and Separation

In some embodiments, the electric field and subsequently the Jouleheating within an affinity SCODA gel are controlled by both the voltageapplied to the source electrodes, and the shape of the gel. Marziali etal.¹ used superimposed rotating dipole and quadrupole fields to driveelectrophoretic SCODA concentration. The ratio of the strength of thesetwo fields, the dipole to quadrupole ratio (D/Q), has an impact on theefficiency of SCODA focusing with a maximum at around D/Q=4.5, howeverthe optimum is relatively flat with the SCODA force staying relativelyconstant for values between 1.75 and 10¹³. One convenient choice of D/Qis 2. With this particular choice, only two distinct potentials need tobe applied to the source electrodes, which can be achieved by connectingone electrode to a common voltage rail, grounding the other three, androtating this pattern in a stepwise manner through the four possibleconfigurations as shown in Table 1. Although analog amplifiers can beused and were used in the examples described herein, using a D/Q ratioof 2 allows one to use discrete MOSFET switches, which simplifies andreduces the required size and complexity of the power supplies.

TABLE 1 Voltage pattern for SCODA focusing with D/Q = 2. Electrode AElectrode B Electrode C Electrode D Step 1 −V 0 0 0 Step 2 0 −V 0 0 Step3 0 0 −V 0 Step 4 0 0 0 −V

A starting point for a sequence specific gel geometry was the four-sidedgel geometry used for the initial demonstration of electrophoreticSCODA. This geometry can be defined by two numbers, the gel width andthe corner radius. The inventors started by using a geometry that had awidth of 10 mm and a corner radius of 3 mm. An electro-thermal model ofthis geometry was implemented in COMSOL Multiphysics® modeling software(COMSOL, Inc, Burlington Mass., USA) to estimate the electric field andtemperature profiles within the gel and establish whether or not thosefield and temperature profiles could drive concentration of a targetwith a temperature dependent mobility. The model used simultaneouslysolves Ohm's Law and the heat equation within the domain, using thepower density calculated from the solution of Ohm's Law as the sourceterm for the heat equation and using the temperature solution from theheat equation to determine the temperature dependent electricalconductivity of the electrolyte in the gel.

To obtain an accurate estimate of the temperature profile within thegel, the heat conducted out of the top and bottom of the gel aremodeled. Boundary conditions and other model parameters are illustratedin FIG. 7. The thermal properties of water and electrical properties of0.2 M NaCl were used. The gel cassettes are placed on an aluminumspreader plate that acts as a constant temperature reservoir. To modelheat flow into the spreader plate the heat transfer coefficient of theglass bottom, given by lilt, was used. The temperature and electricfield profiles solved by this model for a single step of the SCODA cycleare shown in FIG. 8. The voltage applied to the four electrodes was −120V, 0 V, 0 V, 0 V, and the spreader plate temperature was set to 55° C.(328 K). The colour map indicates gel temperature and the vector fieldshows the relative magnitude and direction of the electric field withinthe gel. Note that as DNA is negatively charged its migration directionwill be opposite to the direction of the electric field.

Using experimentally determined values of mobility versus temperaturefor a given molecule and the thermal model described above, it ispossible to determine the SCODA velocity everywhere in the gel for thatparticular molecule by taking the time average of the instantaneousdrift velocity integrated over one complete cycle:

$\begin{matrix}{{\overset{->}{v}}_{s} = {\frac{1}{\tau}{\int_{0}^{\tau}{{\mu \left( {T\left( {\overset{->}{r},t} \right)} \right)}{\overset{->}{E}\left( {\overset{->}{r},t} \right)}{t}}}}} & \lbrack 33\rbrack\end{matrix}$

where μ is the temperature dependent mobility, E the electric field andτ the period of the SCODA cycle. The temperature and electric field weresolved for four steps in the SCODA cycle and coupled with the mobilityfunction in equation [23]. In this manner, the SCODA velocity everywherein the gel can be calculated. Since discrete steps are being used, if itis assumed that the period is long enough that the phase lag between theelectric field and temperature can be neglected, then the integral inequation [33] becomes a sum:

$\begin{matrix}{{\overset{->}{v}}_{s} = \frac{\sum{{\mu \left( {T_{i}\left( \overset{->}{r} \right)} \right)}{{\overset{->}{E}}_{i}\left( \overset{->}{r} \right)}t_{i}}}{\sum t_{i}}} & \lbrack 34\rbrack\end{matrix}$

where the velocity is summed over all four steps in the cycle.

As an example, FIG. 9 shows a vector plot of the SCODA velocity usingthe experimentally determined mobility versus temperature curve for theperfect match target shown in FIG. 11 (example described below) and thetemperature and electric field values calculated above.

The velocity field plotted in FIG. 9 shows a zero velocity point at thegeometric centre of the gel, with the velocity at all other points inthe gel pointing towards the centre. Thus, target molecules can becollected within the gel at the centre of the electric field pattern.

In embodiments that are used to separate two similar molecules based ondifferences in binding affinity for the immobilized probe, a washingforce is superimposed over the SCODA focusing fields described above. Insome embodiments, the washing force is a DC electric field, describedherein as a DC bias. For molecules having affinity to the immobilizedprobe, the SCODA focusing force applied by the SCODA focusing fieldsdescribed above will tend to counteract movement of a molecule caused bythe washing field, i.e. the SCODA focusing fields will tend to exert arestoring force on the molecules and the molecules will bepreferentially focused as compared with molecules having a smallerbinding affinity. Molecules that have a smaller binding affinity to theimmobilized probe will have a greater mobility through the affinitymatrix, and the restoring SCODA force will be weaker. As a result, thefocus spot of molecules with a smaller binding affinity will be shifted.In some cases, the restoring SCODA force will be so weak that suchmolecules with a smaller binding affinity will be washed out of theaffinity matrix altogether.

In order to enrich for a specific biomolecule from a population of othersimilar biomolecules using affinity SCODA, one may operate SCODAfocusing electric fields with a superimposed DC bias. The DC bias maymove the focused molecules off centre, in such a way that the moleculeswith a lower binding energy to the immobilized binding sites movefurther off centre than the molecules with higher binding energies, thuscausing the focus to split into multiple foci. For molecules withsimilar binding energies, this split may be small while washing underbias. The DC bias may be superimposed directly over the focusing fields,or a DC field may be time multiplexed with the focusing fields.

In one exemplary embodiment used to separate nucleic acids havingsimilar sequences, a DC bias is superimposed over the voltage patternshown in Table 1, resulting in the voltage pattern shown below in Table2. In some embodiments, the DC bias is applied alternately with theSCODA focusing fields, i.e. the SCODA focusing fields are applied for aperiod of time then stopped, and the DC bias is applied for a period oftime then stopped.

TABLE 2 Applied voltages for focusing under a DC bias. Shown are valuesfor a 120 V SCODA focusing potential superimposed over a 10 V DC bias.Electrode A Electrode B Electrode C Electrode D Step 1 −120 5 10 5 Step2 0 −115 10 5 Step 3 0 5 −110 5 Step 4 0 5 10 −115

The resulting velocity plots of both the perfect match and single basemismatch targets in the presence of the applied DC bias are shown inFIGS. 10A and 10B, respectively. Electric field and temperature werecalculated using COMSOL using a spreader plate temperature of 61° C.Velocity was calculated using equation [34] and the experimentallyobtained data fits shown in FIG. 11 (example described below). The zerovelocity location of the perfect match target has been moved slightlyoff centre in the direction of the bias (indicated with a circularspot), however the mismatch target has no zero velocity point within thegel. These calculations show that it is possible to completely wash atarget with a smaller binding affinity from the immobilized probe fromthe gel area while capturing the target with a higher binding affinity,enabling selective purification, concentration and/or detection of aspecific sequence, even where the nucleotide targets differ in sequenceat only one position.

In some embodiments, the optimal combination of the driving field andthe mobility altering field used to perform SCODA focusing where thereis a maximum difference in focusing force between similar molecules isempirically determined by measuring the velocity of sample moleculesthrough a medium as a function of the mobility varying field. Forexample, in some embodiments the mobility of a desired target moleculeand a non-desired target molecule at various temperatures is measured inan affinity matrix as described above, and the temperature range atwhich the difference in relative mobility is greatest is selected as thetemperature range for conducting affinity SCODA. In some embodiments,the focusing force is proportional to the rate at which the velocitychanges with respect to the perturbing field dv/df, where v is themolecule velocity and f the field strength. One skilled in the art maymaximize dv/df so as to maximize SCODA focusing and to enable fastwashing of contaminants that do not focus. To maximally separate twosimilar molecules, affinity SCODA may be carried out under conditionssuch that dv_(a)/df−dv_(b)/df (where v_(a) is the velocity of moleculea, and v_(b) is the velocity of molecule b) is maximized.

In some embodiments, the strength of the electric field applied to anaffinity matrix is calculated so that the highest temperature within thegel corresponds approximately to the temperature at which the differencein binding affinity between two molecules to be separated is highest.

In some embodiments, the temperature at which the difference in bindingaffinity between the two molecules to be separated is highestcorresponds to the temperature at which the difference between themelting temperature of a target molecule and the affinity agent and themelting temperature of a non-target molecule and the affinity agent ishighest. In some embodiments, the maximum difference between the meltingtemperature of a target molecule and the affinity agent and the meltingtemperature of a non-target molecule and the affinity agent is less thanabout 9.3° C., in some embodiments less than about 7.8° C., in someembodiments less than about 5.2° C., and in some embodiments less thanabout 0.7° C.

In some embodiments, the ratio of target molecules to non-targetmolecules that can be separated by affinity SCODA is any ratio from 1:1to 1:10,000 and any value therebetween, e.g. 1:100 or 1:1,000. In someembodiments, after conducting affinity SCODA, the ratio of non-targetmolecules relative to target molecules that is located in a focus spotof the target molecules has been reduced by a factor of up to 10,000fold.

Phase Lag Induced Rotation

In some embodiments, to separate molecules with different affinities forthe immobilized affinity agent, a DC bias is superimposed over the SCODAfocusing fields as described above. If the separation in binding energyis great enough then the mismatched target can be washed entirely off ofthe gel. The ability to wash weakly focusing contaminating fragmentsfrom the gel can be affected by the phase lag induced rotation discussedabove, where the SCODA velocity of a two dimensional system was givenby:

{right arrow over (υ)}_(SCODA)=|υ_(SCODA)|(cos(φ){circumflex over(r)}+sin(φ){circumflex over (θ)})  [35]

where φ is the phase lag between the electric field oscillations and themobility varying oscillations. Aside from reducing the proportion of theSCODA velocity that contributes to concentration this result hasadditional implications when washing weakly focusing contaminants out ofan affinity matrix. The rotational component will add to the DC bias andcan result in zero or low velocity points in the gel that cansignificantly increase the time required to wash mismatched targets fromthe gel.

To counteract the effects of a rotational component of motion that mayarise in embodiments in which there is a phase lag between the electricfield oscillations and the mobility varying oscillations, the directionin which the SCODA focusing fields are applied may be rotatedperiodically. In some embodiments, the direction in which the SCODAfocusing fields are rotated is altered once every period.

Optical Feedback

In some embodiments where one molecule of interest (the target molecule)is concentrated in an affinity matrix while a second, similar, molecule(the non-target molecule) is washed off of the affinity matrix, opticalfeedback may be used to determine when washing is complete and/or toavoid running the target molecule out of the affinity matrix.

The two foci of similar molecules may be close together geographically,and optical feedback may be used to ensure the molecule of interest isnot washed off the gel. For example, using a fluorescent surrogate forthe molecule of interest or the contaminating molecules (or both) onecan monitor their respective positions while focusing under bias, anduse that geographical information to adjust the bias ensuring that themolecule of interest is pushed as close to the edge of the gel aspossible but not off, while the contaminating molecule may be removedfrom the gel.

In some embodiments, the molecules to be separated are differentiallylabeled, e.g. with fluorescent tags of a different colour. Real-timemonitoring using fluorescence detection can be used to determine whenthe non-target molecule has been washed off of the affinity matrix, orto determine when the foci of the target molecule and the non-targetmolecule are sufficiently far apart within the affinity matrix to allowboth foci to be separately extracted from the affinity matrix.

In some embodiments, fluorescent surrogate molecules that focussimilarly to the target and/or non-target molecules may be used toperform optical feedback. By using a fluorescent surrogate for a targetmolecule, a non-target molecule, or both a target molecule and anon-target molecule, the respective positions of the target moleculeand/or the non-target molecule can be monitored while performingaffinity focusing under a washing bias. The location of the surrogatemolecules within the affinity matrix can be used to adjust the washingbias to ensure that the molecule of interest is pushed as close to theedge of the gel as possible but not off, while the contaminatingmolecule may be washed off the gel.

In some embodiments, fluorescent surrogate molecules that focussimilarly to the target and/or non-target molecules but will not amplifyin any subsequent PCR reactions that may be conducted can be added to asample to be purified. The presence of the fluorescent surrogatemolecules within the affinity matrix enables the use of optical feedbackto control SCODA focusing conditions in real time. Fluorescencedetection can be used to visualize the position of the fluorescentsurrogate molecules in the affinity matrix. In embodiments where thefluorescent surrogate mimics the focusing behaviour of the targetmolecule, the applied washing force can be decreased when thefluorescent surrogate approaches the edge of the affinity matrix, toavoid washing the target molecule out of the affinity matrix. Inembodiments where the fluorescent surrogate mimics the focusingbehaviour of the non-target molecule that is to be separated from thetarget molecule, the applied washing force can be decreased or stoppedafter the fluorescent surrogate has been washed out of the affinitymatrix, or alternatively when the location of the fluorescent surrogateapproaches the edge of the affinity matrix.

Separation of Differentially Modified Molecules

In some embodiments, molecules that are identical except for thepresence or absence of a chemical modification that alters the bindingaffinity of the molecule for a probe are separated using affinity SCODA.Some embodiments of affinity SCODA are sufficiently sensitive toseparate two molecules that have only a small difference in bindingaffinity for the immobilized affinity agent. Examples of such moleculesinclude differentially modified molecules, such as methylated andunmethylated nucleic acids, methylated or acetylated proteins, or thelike.

For example, it has been previously shown that methylation of cytosineresidues increases the binding energy of hybridization relative tounmethylated DNA sequences. RNA sequences would be expected to display asimilar increase in the binding energy of hybridization when methylatedas compared with unmethylated sequences. The inventors have shown thatone embodiment of affinity SCODA can be used to separate nucleic acidsequences differing only by the presence of a single methylated cytosineresidue. Other chemical modifications would be expected to alter thebinding energy of a nucleic acid and its complimentary sequence in asimilar manner. Modification of proteins, such as through methylation,can also alter the binding affinity of a protein of interest with aprotein, RNA or DNA aptamer, antibody, or other molecule that binds tothe protein at or near the methylation site. Accordingly, embodiments ofaffinity SCODA can be used to separate differentially modified moleculesof interest. While the examples herein are directed to methylationenrichment, affinity SCODA can also be applied to enrichment andselection of molecules with other chemical differences, including e.g.acetylation.

Affinity SCODA, and sequence-specific SCODA, may be used to enrich aspecific sequence of methylated DNA out of a background of methylatedand unmethylated DNA. In this application of affinity SCODA, thestrength of the SCODA focusing force may be related to the bindingenergy of the target DNA to the bound oligonucleotides. Target moleculeswith a higher binding energy may be made to focus more strongly thantargets with lower binding energy. Methylation of DNA has previouslybeen documented to slightly increase the binding energy of target DNA toits complementary sequence. Small changes in binding energy of acomplementary oligonucleotide may be exploited through affinity SCODA topreferentially enrich for methylated DNA. SCODA operating conditions maybe chosen, for example as described above, such that the methylated DNAis concentrated while unmethylated DNA of the same sequence is washedoff the gel.

Some embodiments can separate molecules with a difference in bindingenergy to an immobilized affinity agent of less than kT, the thermalexcitation energy of the target molecules. Some embodiments can separatemolecules with a difference in binding energy to an immobilized affinityagent of less than 0.19 kcal/mol. Some embodiments can separatemolecules with a difference in binding energy to an immobilized affinityagent of less than 2.6 kcal/mol. Some embodiments can separate moleculeswith a difference in binding energy to an immobilized affinity agent ofless than 3.8 kcal/mol. Some embodiments can separate molecules thatdiffer only by the presence of a methyl group. Some embodiments canseparate nucleic acid sequences that differ in sequence at only onebase.

Applications of Affinity SCODA

Systems and methods for separating, purifying, concentrating and/ordetecting differentially modified molecules as described above can beapplied in fields where detection of biomarkers, specific nucleotidesequences or differentially modified molecules is important, e.g.epigenetics, fetal DNA detection, pathogen detection, cancer screeningand monitoring, detection of organ failure, detection of various diseasestates, and the like. For example, in some embodiments affinity SCODA isused to separate, purify, concentrate and/or detect differentiallymethylated DNA in such fields as fetal diagnostic tests utilizingmaternal body fluids, pathogen detection in body fluids, and biomarkerdetection in body fluids for detecting cancer, organ failure, or otherdisease states and for monitoring the progression or treatment of suchconditions.

In some embodiments, a sample of bodily fluid or a tissue sample isobtained from a subject. Cells may be lysed, genomic DNA is sheared, andthe sample is subjected to affinity SCODA. In some embodiments,molecules concentrated using affinity SCODA are subjected to furtheranalysis, e.g. DNA sequencing, digital PCR, fluorescence detection, orthe like, to assay for the presence of a particular biomarker ornucleotide sequence. In some embodiments, the subject is a human.

It is known that fetal DNA is present in maternal plasma, and thatdifferential methylation of maternal versus fetal DNA obtained from thematernal plasma can be used to screen for genetic disorders (see e.g.Poon et al., 2002, Clinical Chemistry 48:1, 35-41). However, one problemthat is difficult to overcome is discrimination between fetal andmaternal DNA. Affinity SCODA as described above may be used topreferentially separate, purify, concentrate and/or detect DNA which isdifferentially methylated in fetal DNA versus maternal DNA. For example,affinity SCODA may be used to concentrate or detect DNA which ismethylated in the fetal DNA, but not in maternal DNA, or which ismethylated in maternal DNA but not fetal DNA. In some embodiments, asample of maternal plasma is obtained from a subject. and subjected toaffinity SCODA using an oligonucleotide probe directed to a sequence ofinterest. The detection of two foci after the application of SCODAfocusing fields may indicate the presence of DNA which is differentiallymethylated as between the subject and the fetus. Comparison to areference sample from a subject that exhibits a particular geneticdisorder may be used to determine if the fetus may be at risk of havingthe genetic disorder. Further analysis of the sample of DNA obtainedthrough differential modification SCODA through conventional methodssuch as PCR, DNA sequencing, digital PCR, fluorescence detection, or thelike, may be used to assess the risk that the fetus may have a geneticdisorder.

One embodiment of the present systems and methods is used to detectabnormalities in fetal DNA, including chromosome copy numberabnormalities. Regions of different chromosomes that are known to bedifferentially methylated in fetal DNA as opposed to maternal DNA areconcentrated using affinity SCODA to separate fetal DNA from maternalDNA based on the differential methylation of the fetal DNA in a maternalplasma sample. Further analysis of the separated fetal DNA is conducted(for example using qPCR, DNA sequencing, fluorescent detection, or othersuitable method) to count the number of copies from each chromosome anddetermine copy number abnormalities.

Most cancers are a result of a combination of genetic changes andepigenetic changes, such as changes in DNA methylation (e.g.hypomethylation and/or hypermethylation of certain regions, see e.g.Ehrich, 2002, Oncogene 21:35, 5400-5413). Affinity SCODA can be used toseparate, purify, concentrate and/or detect DNA sequences of interest toscreen for oncogenes which are abnormally methylated. Embodiments ofaffinity SCODA are used in the detection of biomarkers involving DNAhaving a different methylation pattern in cancerous or pre-cancerouscells than in healthy cells. Detection of such biomarkers may be usefulin both early cancer screening, and in the monitoring of cancerdevelopment or treatment progress. In some embodiments, a sampleobtained from a subject, e.g. a sample of a bodily fluid such as plasmaor a biopsy, may be processed and analyzed by differential modificationSCODA using oligonucleotide probes directed to a sequence of interest.The presence of two foci during the application of SCODA fields mayindicate the presence of differential methylation at the DNA sequence ofinterest. Comparison of the sample obtained from the subject with areference sample (e.g. a sample from a healthy patient and/or a sampleknown to originate from cancerous or pre-cancerous tissue) can indicatewhether the cells of the subject are at risk of being cancerous orpre-cancerous. Further analysis of the sample of DNA obtained throughdifferential modification SCODA through conventional methods such asPCR, DNA sequencing, digital PCR, fluorescence detection, or the like,may be used to assess the risk that the sample includes cells that maybe cancerous or pre-cancerous, to assess the progression of a cancer, orto assess the effectiveness of treatment.

In some embodiments, a specific nucleotide sequence is captured in thegel regardless of methylation (i.e. without selecting for a particularmethylation status of the nucleic acid). Undesired nucleotide sequencesand/or other contaminants may be washed off the gel while the specificnucleotide sequence remains bound by oligonucleotide probes immobilizedwithin the separation medium. Then, differential methylation SCODA isused to focus the methylated version of the sequence while electricallywashing the unmethylated sequence toward a buffer chamber or another gelwhere it can then be recovered. In some embodiments, the unmethylatedsequence could be preferentially extracted.

In some embodiments, biomolecules in blood related to disease states orinfection are selectively concentrated using affinity SCODA. In someembodiments, the biomolecules are unique nucleic acids with sequence orchemical differences that render them useful biomarkers of diseasestates or infection. Following such concentration, the biomarkers can bedetected using PCR, sequencing, or similar means. In some embodiments, asample of bodily fluid or tissue is obtained from a subject, cells arelysed, genomic DNA is sheared, and affinity SCODA is performed usingoligonucleotide probes that are complimentary to a sequence of interest.Affinity SCODA is used to detect the presence of differentiallymethylated populations of the nucleic acid sequence of interest. Thepresence of differentially methylated populations of the target sequenceof interest may indicate a likelihood that the subject suffers from aparticular disease state or an infection.

In some embodiments, the focusing pattern of the target nucleic acidproduced by affinity SCODA from a subject is compared with the focusingpattern of the target nucleic acid produced by affinity SCODA from oneor more reference samples (e.g. an equivalent sample obtained from ahealthy subject, and/or an equivalent sample obtained from a subjectknown to be suffering from a particular disease). Similarities betweenthe focusing pattern produced by the sample obtained from the subjectand a reference sample obtained from a subject known to be sufferingfrom a particular disease indicate a likelihood that the subject issuffering from the same disease. Differences between the focusingpattern produced from the sample obtained from the subject and areference sample obtained from a healthy subject indicate a likelihoodthat the subject may be suffering from a disease. Differences in thefocusing pattern produced from the sample obtained from the subject anda reference sample obtained from a healthy subject may indicate thepresence of a differential modification or a mutation in the subject ascompared with the healthy subject.

Use of Multiple Affinity Agents to Capture Multiple Target Molecules

In some embodiments, affinity SCODA is used to separate, purify,concentrate and/or detect more than one sequence per sample. Theexamples described herein demonstrate that it is possible to concentratetarget DNA at probe concentrations as low as 1 μM, as well as with probeconcentrations as high as 100 μM. In some embodiments, multiplexedconcentration is be performed by immobilizing a plurality of differentaffinity agents in the medium to provide an affinity matrix. In someembodiments, at least two different affinity agents are immobilizedwithin a medium to separate, purify, concentrate and/or detect at leasttwo different target molecules. In some embodiments, each one of theaffinity agents is an oligonucleotide probe with a different sequence.In some embodiments, anywhere between 2 and 100 differentoligonucleotide probes are immobilized within a medium to provide anaffinity matrix, and any where between 2 and 100 different targetmolecules are separated, purified, concentrated and/or detectsimultaneously in a single affinity gel. Each one of the targetmolecules may be labeled with a different tag to facilitate detection,for example each one of the target molecules could be labeled with adifferent colour of fluorescent tag.

In some embodiments where the binding energy between each of the two ormore affinity agents and the two or more target molecules differs, thetwo or more target molecules may be differentially separated within theaffinity matrix by the application of SCODA focusing fields at anappropriate temperature. In some embodiments, a first target moleculewith a lower melting temperature for its corresponding affinity agentmay be preferentially separated from a second target molecule with arelatively higher melting temperature for its corresponding affinityagent. In some such embodiments, the first molecule is preferentiallyconcentrated by conducting SCODA focusing at a temperature that issufficiently low that a second target molecule with a relatively highermelting temperature for its corresponding affinity agent does not focusefficiently (i.e. a temperature at which the mobility of the secondtarget molecule within the affinity matrix is relatively low), butsufficiently high to enable efficient focusing of the first molecule. Insome such embodiments, the first and second molecules are differentiallyseparated through the application of a washing bias, e.g. a DC bias, ata temperature that is sufficiently low that the second target moleculeis not displaced or is displaced only slowly by the washing bias, butsufficiently high that the first target molecule is displaced or isdisplaced at a higher velocity by the washing bias.

Apparatus for Performing Affinity SCODA

In some embodiments, affinity SCODA is performed on an electrophoresisapparatus comprising a region for containing the affinity matrix, bufferreservoirs, power supplies capable of delivering large enough voltagesand currents to cause the desired effect, precise temperature control ofthe SCODA medium (which is a gel in some embodiments), and a two colourfluorescence imaging system for the monitoring of two differentmolecules in the SCODA medium.

EXAMPLES

Embodiments of the invention are further described with reference to thefollowing examples, which are intended to be illustrative and notrestrictive in nature. Although the examples below are described withreference to the separation of DNA oligonucleotides and methylated DNAoligonucleotides, embodiments of the present invention also haveapplication in the purification and separation of other molecules havingan affinity for agents immobilized within a medium, including otherdifferentially modified molecules. Examples of such molecules includepolypeptides or proteins, differentially modified polypeptides orproteins, differentially modified nucleic acids including differentiallymethylated DNA or RNA, or the like. Examples of agents that can beimmobilized as probes in embodiments of the invention include DNA, RNA,antibodies, polypeptides, proteins, nucleic acid aptamers, and otheragents with affinity for a molecule of interest.

Example 1.0 Affinity SCODA with Single Base Mismatch

To verify the predicted temperature dependent mobility expressed inequation [23], experiments were performed to measure the response oftarget DNA velocity to changes in temperature. Initial experiments weredone with 100 nucleotide oligonucleotides as target DNA.Oligonucleotides are single stranded so do not need to be denatured tointeract with the affinity gel. The oligonucleotides are alsosufficiently short that they have a negligible field dependent mobility.Longer nucleic acid molecules, e.g. greater than about 1000 nucleotidesin length, may be difficult to separate based on sequence, as longermolecules have a tendency to focus in a non-sequence specific mannerfrom the electrophoretic SCODA effect in embodiments using Joule heatingprovided by an electric field to provide the temperature gradient.

To perform these measurements a polyacrylamide gel (4% T, 2% C) in 1×TB(89 mM tris, 89 mM boric acid) with 0.2 M NaCl and 10 μM acrydite probe(SEQ ID NO. 1) oligo was cast in a one dimensional gel cassettecontaining only two access ports. Polymerization was initiated throughthe addition of 2 μl of 10% w/v APS and 0.2 μl TEMED per ml of gel.

Mobility measurements were performed on two different 100 nucleotideoligonucleotides differing by a single base containing sequences with aperfect match (PM) (SEQ ID NO. 2) to the probe and a single basemismatch (sbMM) (SEQ ID NO. 3). These target oligonucleotides were endlabeled with either 6-FAM or Cy5 (IDT DNA). Probe and target sequencesused for these experiments are shown in Table 3. The regions of the PMand sbMM target oligonucleotides that are complementary to theimmobilized probe are shown in darker typeface than the other portionsof these oligonucleotides. The position of the single base mismatch isunderlined in the sbMM target sequence.

TABLE 3 Probe and target oligonucleotide sequencesused for sequence specific SCODA. Sequence Probe 5′ACT GGC CGT CGT TTT ACT 3′ (SEQ ID NO.: 1) PM Target 5′CGA TTA AGT TGA GTA ACG CCA CTA TTT (SEQ IDTCA CAG TCA TAA CCA TGT AAA ACG ACG NO.: 2)GCC AGT GAA TTA GCG ATG CAT ACC TTG GGA TCC TCT AGA ATG TAC C 3′sbMM Target 5′ CGA TTA AGT TGA GTA ACG CCA CTA (SEQ IDTTT TCA CAG TCA TAA CCA TGT AAA AC T NO.: 3)ACG GCC AGT GAA TTA GCG ATG CAT ACC TTG GGA TCC TCT AGA ATG TAC C 3′

The probe sequence was chosen to be complementary to pUC19 forsubsequent experiments with longer targets, discussed below. The 100nucleotide targets contain a sequence complementary to the probe(perfect match: PM) or with a single base mismatch (sbMM) to the probewith flanking sequences to make up the 100 nucleotide length. Theflanking sequences were designed to minimize the effects of secondarystructure and self hybridization. Initial sequences for the regionsflanking the probe binding site were chosen at random. Folding and selfhybridization energies were then calculated using Mfold¹⁴ and thesequences were altered one base at a time to minimize these effectsensuring that the dominant interactions would be between target strandsand the probe.

Table 4 shows the binding energies and melting temperatures for thesequences shown in Table 3 calculated using Mfold. The binding energy,ΔG, is given as ΔH−TΔS, where ΔH is the enthalpy and ΔS the entropy inunits of kcal/mol and kcal/mol K respectively. The following parametervalues were used for calculation of the values in Table 2:temperature=50° C., [Na+]=0.2 M, [Mg++]=0 M, strand concentration=10 μM.The largest T. for non probe-target hybridization is 23.9° C. and thegreatest secondary structure T. is 38.1° C. Both of these values are farenough below the sbMM target-probe T_(m) that they are not expected tointerfere target-probe interactions.

TABLE 4 Binding energies and melting temperatures for Table 3 sequences.Probe PM Target sbMM Target Secondary (SEQ ID NO.: 1) (SEQ ID NO.: 2)(SEQ ID NO.: 3) Structure Probe −35.4 + 0.1012 * T −145.3 + 0.4039 * T−126.8 + 0.3598 * T −20.3 + 0.07049 * T (SEQ ID Tm = 12.2° C. Tm = 65.1°C. Tm = 55.8° C. Tm = 14.8° C. NO.: 1) PM Target −145.3 + 0.4039 * T−40.2 + 0.1124 * T −40.2 + 0.1111 * T −24.3 + 0.07808 * T (SEQ ID Tm =65.1° C. Tm = 23.9° C. Tm = 20.9° C. Tm = 38.1° C. NO.: 2) sbMM −126.8 +0.3598 * T −40.2 + 0.1111 * T −40.2 + 0.1124 * T −24.3 + 0.07808 * TTarget Tm = 55.8° C. Tm = 20.9° C. Tm = 23.9° C. Tm = 38.1° C. (SEQ IDNO.: 3)

To measure the velocity response as a function of temperature thefluorescently labeled target was first injected into the gel at hightemperature (70° C.), and driven under a constant electric field intothe imaging area of the gel. Once the injected band was visible thetemperature of the spreader plate was dropped to 55° C. An electricfield of 25 V/cm was applied to the gel cassette while the temperaturewas ramped from 40° C. to 70° C. at a rate of 0.5° C./min. Images of thegel were taken every 20 sec. Image processing software written inLabView® (National Instruments, Austin Tex.) was used to determine thelocation of the centre of the band in each image and this position datawas then used to calculate velocity.

FIG. 11 shows a plot of target DNA mobility as a function oftemperature. Using the values of ΔG for the probe and target sequencesshown in Table 3, the velocity versus temperature curves were fit toequation [23] to determine the two free parameters: the mobility μ₀, andβ a constant that depends on the kinetics of the hybridization reaction.

A fit of the data shown in FIG. 11 shows good agreement with the theoryof migration presented above. Data for the mismatch mobility are shownas the curve on the left, and data for the perfect match mobility areshown as the curve on the right. The R² value for the PM fit and MM fitswere 0.99551 and 0.99539 respectively. The separation between theperfect match and single base mismatch targets supports that there is anoperating temperature where the focusing speed of the perfect matchtarget is significantly greater than that of the mismatched targetenabling separation of the two species through application of a DC biasfield as illustrated in FIG. 4.

Example 2.0 Selective Separation of Molecules Using Affinity SCODA

A 4% polyacrylamide gel containing 10 μM acrydite modified probe oligos(Integrated DNA Technologies, www.idtdna.com) was cast in a gel cassetteto provide an affinity matrix.

Equimolar amounts of the perfect match and single base mismatch targetswere injected into the affinity gel at 30° C. with an electric field of100 V/cm applied across the gel such that both target molecules would beinitially captured and immobilized at the gel buffer interface. Thetemperature was then increased to 70° C. and a constant electric fieldof 20 V/cm applied to the gel to move the target into the imaging areaof the gel. The temperature was then dropped to 62° C. and a 108 V/cmSCODA focusing field superimposed over an 8 V/cm DC bias as shown inTable 2 was applied to the four source electrodes with a period of 5seconds. The rotation direction of the SCODA focusing field was alteredevery period.

TABLE 5 Focusing plus bias potentials applied. Electrode A Electrode BElectrode C Electrode D Step 1 −108 4 8 4 Step 2 0 −104 8 4 Step 3 0 4−100 4 Step 4 0 4 8 −104

FIG. 12 shows images of concentration taken every 2 minutes. The perfectmatch target was tagged with 6-FAM and shown in green (leading brightspot which focuses to a tight spot), the mismatch target was tagged withCy5 and is shown in red (trailing bright line that is washed from thegel). The camera gain was reduced on the green channel after the firstimage was taken. DNA was injected into the right side of the gel andfocusing plus bias fields were applied. The perfect match target (green)experiences a drift velocity similar to that shown in FIG. 10A and movestowards a central focus location. The more weakly focusing mismatchtarget (red) experiences a velocity field similar to that shown in FIG.10B and is pushed off the edge of the gel by the bias field. Thedirection of application of the applied washing field is indicated bythe white arrow.

This experiment verifies the predictions of FIGS. 10A and 10Bdemonstrating that it is possible to generate two different velocityprofiles for two DNA targets differing by only a single base enablingpreferential focusing of the target with the higher binding energy tothe gel. The images in FIG. 12 confirm that there are two distinctvelocity profiles generated for the two different sequences of targetDNA moving through an affinity matrix under the application of both aSCODA focusing field and a DC bias. A dispersive velocity field isgenerated for the single base mismatch target and a non dispersivevelocity field is generated for the perfect match target. This exampledemonstrates that it is possible to efficiently enrich for targets withsingle base specificity, and optionally wash a non-desired target off ofan affinity matrix, even if there is a large excess of mismatch targetin the sample.

Example 3.0 Optimization of Operating Conditions

Different parameters of the SCODA process may be optimized to achievegood sample enrichment at reasonable yields. In embodiments havingimmobilized (and negatively charged) DNA in the gel, a relatively highsalinity running buffer was found to provide both efficient and stablefocusing, as well as minimizing the time required to electrokineticallyinject target DNA from an adjacent sample chamber into the SCODA gel.

Example 3.1 Optimization of Buffer Salinity

Early attempts of measuring the temperature dependent mobility ofmolecules in an affinity gel as well as the first demonstrations ofsequence specific SCODA were performed in buffers used forelectrophoretic SCODA. These are typically standard electrophoresisbuffers such as tris-borate EDTA (TBE), often diluted 4 to 6 fold toreduce the gel conductivity, enabling the application of high electricfields within thermal limitations imposed by Joule heating, resulting inshorter concentration times¹³. Although it is possible to achievesequence specific SCODA based concentration in a 1×TBE buffer (89 mMtris, 89 mM boric acid, 2 mM disodium EDTA), conditions can be furtheroptimized for performance of sequence specific SCODA due to therelatively low concentration of dissociated ions at equilibrium in 1×TBEbuffer. A low concentration of dissociated ions results in slowhybridization kinetics, exacerbates ionic depletion associated withimmobilizing charges (oligonucleotide probes) in the gel, and increasesthe time required to electrokinetically inject target DNA into the gel.Calculations using 89 mM tris base and 89 mM boric acid, with a pKa of9.24 for boric acid and a pKa of 8.3 for tris shows a concentration of1.49 mM each of dissociated tris and dissociated boric acid in 1×TBEbuffer.

Example 3.2 Effect of Salt Concentration on DNA Hybridization

In embodiments used to separate nucleic acids, the presence of positivecounter ions shields the electrostatic repulsion of negatively chargedcomplementary strands of nucleic acid, resulting in increased rates ofhybridization. For example, it is known that increasing theconcentration of Na+ ions affects the rate of DNA hybridization in anon-linear manner (see Tsuruoka et al.¹⁵, which is incorporated byreference herein). The hybridization rate increases by about 10 foldwhen [NaCl] is increased from 10 mM to 1 M of [NaCl], with most of thegain achieved by the time one reaches about 200 mM. At lowconcentrations of positive counter ions, below about 10 mM, the rate ofhybridization is more strongly dependent on salt concentration, roughlyproportional to the cube of the salt concentration⁶. Theoreticalcalculations suggest that the total positive counter ion concentrationof 1×TBE is around 5.5 mM (1.5 mM of dissociated tris, and 4 mM of Na+from the disodium EDTA). At this ion concentration it was possible toachieve focusing however the slow hybridization rates resulted in weakfocusing and large final focus spot sizes.

A slow rate of hybridization can lead to weak focusing through anincrease in the phase lag between the changes in electric field andchanges in mobility. Equation [16] describes the SCODA velocity as beingproportional to cos(φ), where φ represents the phase lag between themobility oscillations and the electric field oscillations. In the caseof ssSCODA a phase lag can result from both a slow thermal response aswell as from slow hybridization kinetics.

This phase lag results in slower focusing times and larger spot sizessince the final spot size is a balance between the inward SCODA-drivendrift, and outward diffusion-driven drift. Faster focusing times arealways desirable as this tends to reduce the overall time to enrich atarget from a complex mixture. A smaller spot size is also desirable asit improves the ability to discriminate between different molecularspecies. As discussed above, when performing SCODA focusing underapplication of a DC bias, the final focus spot will be shifted offcenter by an amount that depends on both the mobility of the target andthe speed of focusing, both of which depend on the strength of theinteraction between the target and the gel bound probes. The amount ofseparation required to discriminate between two similar molecules whenfocusing under bias therefore depends on the final focus spot diameter.Smaller spot diameters should improve the ability to discriminatebetween two targets with similar affinity to the gel bound probes.

At the low rates of hybridization achieved with 1×TBE buffer, reliablefocusing was only achievable with probe concentrations near 100 μM.Increasing the salt concentration from around 5 mM to 200 mM through theaddition of NaCl, while keeping the probe concentration at 100 μM hadthe effect of reducing the final focus spot size as shown in FIGS.13A-D. All images in FIGS. 13A-D were taken after a similar amount offocusing time (approximately 5 min), however the increased salinityresulted in increased Joule heating, which required a four foldreduction of field strength to prevent boiling when focusing with 200 mMNaCl. Probe concentrations are 100 μM, 10 μM, 1 μM, and 100 μM,respectively in FIGS. 13A, 13B, 13C and 13D. The buffer used in FIGS.13A, 13B and 13C was 1×TB with 0.2 M NaCl. The buffer used in FIG. 13Dwas 1×TBE. Focusing was not reliable at 10 μM and 1 μM probe in 1×TBEand these results are not shown. Under equivalent conditions in thisexample, addition of 200 mM NaCl to the gel also allowed for focusing ofcomplementary targets at 100 fold lower probe concentrations.

Equation [30] states that the focusing speed is proportional to theelectric field strength, so that fact that comparable focusing times areachieved with a four fold reduction in electric field strength suggeststhat the field normalized focusing speed is considerably faster underhigh salinity conditions.

Although the total time for focusing was not reduced by the addition of200 mM NaCl, focusing at lower electric field strength may be desirablein some embodiments because lower field strength can limit the degree ofnon-specific electrophoretic SCODA that may occur in an affinity matrixin some embodiments. For example, all target nucleic acid molecules willfocus irrespective of their sequence in the affinity gels used forsequence specific SCODA in embodiments where the thermal gradient isestablished by an electric field due to electrophoretic SCODA. The speedof electrophoretic SCODA focusing increases with electric field¹³, sodecreasing the field strength will have the effect of reducing thenon-specific SCODA focusing speed, allowing one to wash non-target DNAmolecules from the gel more easily by applying a DC bias.

Example 3.3 Ion Depletion and Bound Charges

The rate at which ions are depleted (or accumulated) at a boundaryincreases as the fraction of charges that are immobile increases. The100 μM probe concentration required to achieve efficient concentrationin 1×TBE results in 2 mM of bound negative charges within the gel when a20 nucleotide probe is used, which is comparable to the total amount ofdissolved negative ions within the gel (around 5.5 mM). This highproportion of bound charge can result in the formation of regions withinthe gel that become depleted of ions when a constant electric field isplaced across the gel¹⁶⁻²° as it is during injection and during SCODAfocusing under DC bias.

A high salinity running buffer can therefore help to minimize many ofthe ion depletion problems associated with immobilizing charges in anssSCODA gel by enabling focusing at lower probe concentrations, as wellas reducing the fraction of bound charges by adding additional freecharges.

Example 3.4 Denaturation of Double Stranded DNA

Target DNA will not interact with the gel immobilized probes unless itis single stranded. The simplest method for generating single strandedDNA from double stranded DNA is to boil samples prior to injection. Onepotential problem with this method is that samples can re-anneal priorto injection reducing the yield of the process, as the re-annealeddouble stranded targets will not interact with the probes and can bewashed off of the gel by the bias field. Theoretical calculations showthat the rate of renaturation of a sample will be proportional to theconcentration of denatured single stranded DNA. Provided targetconcentration and sample salinity are both kept low, renaturation of thesample can be minimized.

To measure the effect of target concentration on renaturation andoverall efficiency, fluorescently labeled double stranded PCR ampliconscomplementary to gel bound probes were diluted into a 250 μl volumecontaining about 2 mM NaCl and denatured by boiling for 5 min followedby cooling in an ice bath for 5 min. The sample was then placed in thesample chamber of a gel cassette, injected into a focusing gel andconcentrated to the centre of the gel. After concentration was completethe fluorescence of the final focus spot was measured, and compared tothe fluorescence of the same quantity of target that was manuallypipetted into the centre of an empty gel cassette. This experiment wasperformed with 100 ng (2×10¹¹ copies) and 10 ng (2×10¹⁰ copies) ofdouble stranded PCR amplicons. The 100 ng sample resulted in a yield of40% and the 10 ng sample resulted in a yield of 80%. This exampleconfirms that lower sample DNA concentration will result in higheryields.

Example 3.5 Phase Lag Induced Rotation

As discussed above, in embodiments in which there is a phase lag betweenthe electric field oscillations and the mobility varying oscillations, arotational component will be added to the velocity of molecules movingthrough the affinity matrix. An example of this problem is shown in FIG.14. The targets shown in FIG. 14 focus weakly under SCODA fields andwhen a small bias is applied to wash them from the gel, the wash fieldand the rotational velocity induced by the SCODA fields sum to zero nearthe bottom left corner of the gel. This results in long wash times, andin extreme cases weak trapping of the contaminant fragments. Thedirection of rotation of the electric field used to produce SCODAfocusing is indicated by arrow 34. The direction of the applied washingforce is indicated by arrow 36.

To overcome this problem the direction of the field rotation can bealtered periodically. In other examples described herein, the directionof the field rotation was altered every period. This results in muchcleaner washing and focusing with minimal dead zones. This scheme wasapplied during focus and wash demonstrations described above and shownin FIG. 12, an example in which the mismatched target was cleanly washedfrom the gel without rotation. Under these conditions there is a reducedSCODA focusing velocity due to the phase lag, but there is not anadditional rotational component of the SCODA velocity.

Example 3.6 Effect of Secondary Structure

Secondary structure in the target DNA will decrease the rate ofhybridization of the target to the immobilized probes. This will havethe effect of reducing the focusing speed by increasing the phase lagdescribed in equation [16]. The amount by which secondary structuredecreases the hybridization rate depends on the details of the secondarystructure. With a simple hairpin for example, both the length of thestem and the loop affect the hybridization rate⁹. For most practicalapplications of sequence specific SCODA, where one desires to enrich fora target molecule differing by a single base from contaminatingbackground DNA, both target and background will have similar secondarystructure. In this case the ability to discriminate between target andbackground will not be affected, only the overall process time. Byincreasing the immobilized probe concentration and the electric fieldrotation period one can compensate for the reduced hybridization rate.

There are potentially cases where secondary structure can have an impacton the ability to discriminate a target molecule from backgroundmolecules. It is possible for a single base difference between targetand background to affect the secondary structure in such a way thatbackground DNA has reduced secondary structure and increasedhybridization rates compared to the target, and is the basis for singlestranded conformation polymorphism (SSCP) mutation analysis. This effecthas the potential to both reduce or enhance the ability to successfullyenrich for target DNA, and care must be taken when designing target andprobe sequences to minimize the effects of secondary structure. Once atarget molecule has been chosen, the probe position can be moved aroundthe mutation site. The length of the probe molecule can be adjusted. Insome cases, oligonucleotides can be hybridized to sequences flanking theregion where the probe anneals to further suppress secondary structure.

Example 4.0 Quantitation of Sequence Specific SCODA Performance

The length dependence of the final focus location while focusing underDC bias was measured and shown to be independent of length for fragmentsranging from 200 nt to 1000 nt in length; an important result, whichimplies that ssSCODA is capable of distinguishing nucleic acid targetsby sequence alone without the need for ensuring that all targets are ofa similar length. Measurements confirmed the ability to enrich fortarget sequences while rejecting contaminating sequences differing fromthe target by only a single base, and the ability to enrich for targetDNA that differs only by a single methylated cytosine residue withrespect to contaminating background DNA molecules.

Example 4.1 Length Independence of Focusing

The ability to purify nucleic acids based on sequence alone,irrespective of fragment length, eliminates the need to ensure that alltarget fragments are of similar length prior to enrichment. The theoryof sequence specific SCODA presented above predicts that sequencespecific SCODA enrichment should be independent of target length.However, effects not modeled above may lead to length dependence, andexperiments were therefore performed to confirm the length independenceof sequence specific SCODA.

According to the theory of thermally driven sequence specific SCODAdeveloped above, the final focus location under bias should not dependon the length of the target strands. Length dependence of the finalfocus location enters into this expression through the length dependenceof the unimpeded mobility of the target μ₀. However, since both μ(T_(m))and a are proportional to μ₀, the length dependence will cancel fromthis expression. The final focus location of a target concentrated withthermally driven ssSCODA should therefore not depend on the length ofthe target, even if a bias is present.

There are two potential sources of length dependence in the final focuslocation, not modeled above, which must also be considered:electrophoretic SCODA in embodiments where the temperature gradient isestablished by an electric field, and force based dissociation of probetarget duplexes. DNA targets of sufficient length (>200 nucleotides)have a field dependent mobility in the polyacrylamide gels used forsequence specific SCODA, and will therefore experience a sequenceindependent focusing force when focusing fields are applied to the gel.The total focusing force experienced by a target molecule will thereforebe the sum of the contributions from electrophoretic SCODA and sequencespecific SCODA. Under electrophoretic SCODA, the focusing velocity tendsto increase for longer molecules¹³, while the DC velocity tends todecrease so that under bias the final focus location depends on length.The second potential source of length dependence in the final focuslocation is force based dissociation. The theory of affinity SCODApresented above assumed that probe-target dissociation was drivenexclusively by thermal excitations. However it is possible to dissociatedouble stranded DNA with an applied force. Specifically, an externalelectric field pulling on the charged backbone of the target strand canbe used to dissociate the probe-target duplex. The applied electricfield will tend to reduce the free energy term ΔG in equation [22] by anamount equal to the energy gained by the charged molecule moving throughthe electric field¹². This force will be proportional to the length ofthe target DNA as there will be more charges present for the electricfield to pull on for longer target molecules, so for a given electricfield strength the rate of dissociation should increase with the lengthof the target.

To measure whether or not these two effects contribute significantly tothe length dependence of the final focus location, two different lengthsof target DNA, each containing a sequence complementary to gelimmobilized probes, were focused under bias and the final focus locationmeasured and compared. The target DNA was created by PCR amplificationof a region of pUC19 that contains a sequence complementary to the probesequence in Table 3. Two reactions were performed with a common forwardprimer, and reverse primers were chosen to generate a 250 bp ampliconand a 1000 bp amplicon. The forward primers were fluorescently labeledwith 6-FAM and Cy5 for the 250 bp and 1000 bp fragments respectively.The targets were injected into an affinity gel and focused to the centrebefore applying a bias field. A bias field of 10 V/cm was superimposedover 120 V/cm focusing fields for 10 min at which point the bias wasincreased to 20 V/cm for an additional 7 min. Images of the gel weretaken every 20 sec, with a 1 sec delay between the 6-FAM channel and theCy5 channel. The field rotation period was 5 sec. Images were postprocessed to determine the focus location of each fragment. FIGS. 15Aand 15B show the focus location versus time for the 250 bp (green) and1000 bp (red) fragments. FIG. 15B is an image of final focus of the twofragments at the end of the experiment.

There is a small difference in final location that can be attributed tothe fact that the two images were not taken at the same phase in theSCODA cycle. This example shows that the final focus position does notdepend on length. Thus, under these operating conditions electrophoreticSCODA focusing is much weaker than affinity SCODA focusing, and thataffinity SCODA is driven largely by thermal dissociation rather thanforce-based dissociation. This result confirms that affinity SCODA iscapable of distinguishing nucleic acid targets by sequence alone withoutthe need for ensuring that all targets are of a similar length.

Example 4.2 Single Base Mismatch Rejection Ratio

To demonstrate the specificity of ssSCODA with respect to rejection ofsequences differing by a single base, different ratios of synthetic 100nt target DNA containing either a perfect match (PM) or single basemismatch (sbMM) to a gel bound probe, were injected into an affinitygel. SCODA focusing in the presence of DC wash fields was performed toremove the excess sbMM DNA. The PM target sequence was labeled with6-FAM and the sbMM with Cy5; after washing the sbMM target from the gelthe amount of fluorescence at the focus location was quantified for eachdye and compared to a calibration run. For the calibration run,equimolar amounts of 6-FAM labeled PM and Cy5 labeled PM target DNA werefocused to the centre of the gel and the fluorescence signal at thefocus location was quantified on each channel. The ratio of the signalCy5 channel to the signal on the 6-FAM channel measured during thiscalibration is therefore the signal ratio when the two dye molecules arepresent in equimolar concentrations. By comparing the fluorescenceratios after washing excess sbMM from the gel to the calibration run itwas possible to determine the amount of sbMM DNA rejected from the gelby washing.

Samples containing target sequences shown in Table 3 were added to thesample chamber and an electric field of 50 V/cm was applied across thesample chamber at 45° C. to inject the sample into a gel containing 10μM of immobilized probe. Once the sample was injected into the gel, theliquid in the sample chamber was replaced with clean buffer and SCODAfocusing was performed with a superimposed DC wash field. A focusingfield of 60 V/cm was combined with a DC wash field of 7 V/cm, the latterapplied in the direction opposite to the injection field. It was foundthat this direction for the wash field led to complete rejection of themismatched target DNA in the shortest amount of time. Table 6 belowshows the amount of DNA injected into the gel for each experiment.

TABLE 6 List of targets run for measuring the rejection ratio ofaffinity SCODA with respect to single base differences. Run Description:Cy5 Labeled Target 6-FAM Labeled Target     1:1 Calibration  10 fmol PM10 fmol PM    100:1  1 pmol sbMM 10 fmol PM  1,000:1  10 pmol sbMM 10fmol PM  10,000:1 100 pmol sbMM 10 fmol PM 100,000:1  1 nmol sbMM 10fmol PM

After the mismatched target had been washed from the gel, the focusingfields were turned off and the temperature of the gel was reduced to 25°C. prior to taking an image of the gel for quantification. It wasimportant to ensure that all images used for quantification were takenat the same temperature, since Cy5 fluorescence is highly temperaturedependent, with the fluorescence decreasing at higher temperatures. Theratio of fluorescence on the Cy5 and 6-FAM channels were compared to the1:1 calibration run to determine the rejection ratio for each run. FIGS.16A and 16B show the results of these experiments. Four different ratiosof sbMM:PM were injected into a gel and focused under bias to removeexcess sbMM. The PM DNA was tagged with 6-FAM and the sbMM DNA wastagged with Cy5. FIG. 16A shows the fluorescence signal from the finalfocus spot after excess sbMM DNA had been washed from the gel. Thefluorescence signals are normalized to the fluorescence measured on aninitial calibration run where a 1:1 ratio of PM-FAM:PMCy5 DNA wasinjected and focused to the centre of the gel. FIG. 16B shows therejection ratios calculated by dividing the initial ratio of sbMM:PM bythe final ratio after washing.

It was found that rejection ratios of about 10,000 fold are achievable.However it should be noted that images taken during focusing and wash athigh sbMM:PM ratios suggest that there were sbMM molecules with twodistinct velocity profiles. Most of the mismatch target washed cleanlyoff of the gel while a small amount was captured at the focus. Thesefinal focus spots visible on the Cy5 channel likely consisted of Cy5labeled targets that were incorrectly synthesized with the single basesubstitution error that gave them the PM sequence. The 10,000:1rejection ratio measured here corresponds to estimates ofoligonucleotide synthesis error rates with respect to single basesubstitutions²¹, meaning that the mismatch molecule synthesized by IDTlikely contains approximately 1 part in 10,000 perfect match molecules.This implies that the residual fluorescence detected on the Cy5 channel,rather than being unresolved mismatch may in fact be Cy5 labeled perfectmatch that has been enriched from the mismatch sample. Consequently therejection ratio of ssSCODA may actually be higher than 10,000:1.

Example 4.3 Mutation Enrichment for Clinically Relevant Mutation

The synthetic oligonucleotides used in the example above were purposelydesigned to maximize the difference in binding energy between theperfect match-probe duplex and the mismatch-probe duplex. The ability ofaffinity SCODA to enrich for biologically relevant sequences has alsobeen demonstrated. In this example, cDNA was isolated from cell linesthat contained either a wild type version of the EZH2 gene or a Y641Nmutant, which has previously been shown to be implicated in B-cellnon-Hodgkin Lymphoma²². 460 bp regions of the EZH2 cDNA that containedthe mutation site were PCR amplified using fluorescent primers in orderto generate fluorescently tagged target molecules that could bevisualized during concentration and washing. The difference in bindingenergy between the mutant-probe duplex and the wild type-probe duplex at60° C. was 2.6 kcal/mol compared to 3.8 kcal/mol for the syntheticoligonucleotides used in the previous examples. This corresponds to amelting temperature difference of 5.2° C. for the mutant compared to thewild type. Table 7 shows the free energy of hybridization and meltingtemperature for the wild type and mutants to the probe sequence.

TABLE 7 Binding energy and melting temperatures of EZH2 targets to thegel bound probe. Target Binding Energy Wild Type −161.9 + 0.4646T Tm =57.1° C. Y641N Mutant −175.2 + 0.4966T Tm = 62.3° C.

A 1:1 mixture of the two alleles were mixed together and separated withaffinity SCODA. 30 ng of each target amplicon was added to 300 μl of0.01× sequence specific SCODA running buffer. The target solution wasimmersed in a boiling water bath for 5 min then placed in an ice bathfor 5 min prior to loading onto the gel cassette in order to denaturethe double stranded targets. The sample was injected with an injectioncurrent of 4 mA for 7 min at 55° C. Once injected, a focusing field of150 V/cm with a 10 V/cm DC bias was applied at 55° C. for 20 min.

The result of this experiment is shown in FIGS. 17A, 17B and 17C. Thebehavior of these sequences is qualitatively similar to the higher T_(m)difference sequences shown in the above examples. The wild type(mismatch) target is completely washed from the gel (images on the righthand side of the figure) while the mutant (perfect match) is driventowards the centre of the gel (images on the left hand side of thefigure). In this case the efficiency of focusing was reduced as some ofthe target re-annealed forming double stranded DNA that did not interactwith the gel bound probes.

The lower limit of detection with the optical system used was around 10ng of singly labeled 460 bp DNA.

Example 5.0 Methylation Enrichment

The ability of affinity SCODA based purification to selectively enrichfor molecules with similar binding energies was demonstrated byenriching for methylated DNA in a mixed population of methylated andunmethylated targets with identical sequence.

Fluorescently tagged PM oligonucleotides having the sequence set out inTable 3 (SEQ ID NO. 2) were synthesized by IDT with a single methylatedcytosine residue within the capture probe region (residue 50 in the PMsequence of Table 3). DC mobility measurements of both the methylatedand unmethylated PM strands were performed to generate velocity versustemperature curves as described above; this curve is shown in FIG. 18.

Fitting of these curves to equation [23] suggests that the difference inbinding energy is around 0.19 kcal/mol at 69° C., which is about a thirdof the thermal energy^(FN1). The curve further suggests that separationof the two targets will be most effective at an operating temperature ofaround 69° C., where the two fragments have the greatest difference inmobility as shown in FIG. 19. In this example, the maximum value of thisdifference is at 69.5° C., which is the temperature for maximumseparation while performing SCODA focusing under the application of a DCbias. At 69° C. k_(b)T=0.65 kcal/mol

This temperature is slightly higher than that used in the aboveexamples, and although it should result in better discrimination, focustimes are longer as the higher temperature limits the maximum electricfield strength one can operate at without boiling the gel.

Initial focusing tests showed that it is possible to separate the twotargets by performing affinity SCODA focusing with a superimposed DCbias. FIG. 20 shows the result of an experiment where equimolar ratiosof methylated and unmethylated targets were injected into a gel, focusedwith a period of 5 sec at a focusing field strength of 75 V/cm and abias of 14 V/cm at 69° C. Methylated targets were labeled with 6-FAM(green, spot on right) and unmethylated targets were labeled with Cy5(red, spot on left). The experiment was repeated with the dyes switched,with identical results.

Achieving enrichment by completely washing the unmethylated target fromthe gel proved to be difficult using the same gel geometry for the aboveexamples, as the gel buffer interface was obscured by the buffer wellspreventing the use of visual feedback to control DC bias fields whileattempting to wash the unmethylated target from the gel. To overcomethis problem gels were cast in two steps: first a gel without probeoligonucleotides was cast in one of the arms of the gel and once thefirst gel had polymerized the remainder of the gel area was filled withgel containing probe oligonucleotides. The gels were cast such that theinterface between the two was visible with the fluorescence imagingsystem. This system allowed for real time adjustments in the biasvoltage so that the unmethylated target would enter the gel withoutimmobilized probes and be quickly washed from the gel, while themethylated target could be retained in the focusing gel. FIGS. 21A-21Dshow the result of this experiment. FIGS. 21A and 21B show the resultsof an initial focus before washing unmethylated target from the gel for10 pmol unmethylated DNA (FIG. 21A) and 0.1 pmol methylated DNA (FIG.21B). FIGS. 21C and 21D show the results of a second focusing conductedafter the unmethylated sequence had been washed from the gel forunmethylated and methylated target, respectively. All images were takenwith the same gain and shutter settings.

In this experiment a 100 fold excess of unmethylated target was injectedinto the gel, focused to the centre without any wash fields applied. Thetargets were then focused with a bias field to remove the unmethylatedtarget, and finally focused to the centre of the gel again forfluorescence quantification. Fluorescence quantification of these imagesindicates that the enrichment factor was 102 fold with losses of themethylated target during washing of 20%. This experiment was repeatedwith the dye molecules swapped (methylated Cy5 and unmethylated 6-FAM)with similar results.

Example 6.0 Multiplexed Affinity SCODA

Two different oligonucleotide probes described above, one havingaffinity for EZH2 and one having affinity for pUC, were cast in a gel ata concentration of 10 μM each to provide an affinity matrix containingtwo different immobilized probes. A 100 nucleotide target sequence withaffinity for the EZH2 probe and a theoretical melting temperature of62.3° C. was labeled with Cy5. A 100 nucleotide target sequence withaffinity for the pUC probe and a theoretical melting temperature of70.1° C. was labeled with FAM. The theoretical difference in meltingtemperature between the two target molecules is 7.8° C.

The target molecules were loaded on the affinity gel (FIG. 22A), andfocusing was conducted with the temperature beneath the gel boatmaintained at 55° C. (FIGS. 22B, focusing after two minutes, and 22C,after four minutes). The EZH2 target focused under these conditions(four red spots), while the pUC target focused only weakly under theseconditions (three diffuse green spots visible on the gel). The centralextraction well did not contain buffer during the initial portions ofthis experiment, resulting in the production of four focus spots, ratherthan a single central focus spot. The temperature beneath the gel wasthen increased to 62° C., a temperature increase of 7° C. (FIGS. 22D,focusing two minutes after temperature increase, and 22E, after fourminutes), resulting in the formation of four clear focus spots for thepUC target. The EZH2 target remained focused in four tight spots at thishigher temperature.

The temperature beneath the gel was reduced to 55° C. and buffer wasadded to the central extraction well. Application of SCODA focusingfields at this temperature resulted in the EZH2 target being selectivelyconcentrated into the central extraction well (diffuse red spot visibleat the centre of FIGS. 22F, 0.5 minutes, and 22G, 1 minute) while thepUC target remained largely focused in four spots outside the centralextraction well. The temperature beneath the gel was increased to 62°C., a temperature increase of 7° C. Within two minutes, the pUC targethad been focused into the central extraction well (FIG. 22H, diffuse redand green fluorescence visible at the centre of the gel).

A second experiment was conducted under similar conditions as the first.After focusing the EZH2 target at 55° C. and the pUC target at 62° C. asdescribed above, a DC washing bias was applied to the gel with thetemperature beneath the gel maintained at 55° C. Under these conditions,the EZH2 target experienced a greater bias velocity than the pUC target.The focus spot for the EZH2 target shifted more quickly after theapplication of the bias field (red spot moving to the right of the gelin FIGS. 22I, 6 minutes after application of bias field, 22J, after 12minutes, and 22K, after 18 minutes). The focus spot for the EZH2 targetwas also shifted a farther distance to the right within the gel. Incontrast, the focus spot for the pUC target shifted more slowly (initialgreen focus spots still largely visible in FIG. 22I after 6 minutes,shifting to the right through FIG. 22J, 12 minutes, and 22K, 18minutes), and was not shifted as far to the right as the focus spot forthe EZH2 target by the washing bias.

Affinity SCODA Yield vs Purity

Because affinity SCODA relies on repeated interactions between targetand probe to generate a non-dispersive velocity field for targetmolecules, while generating a dispersive field for contaminants (so longas a washing bias is applied), high specificity can be achieved withoutsacrificing yield. If one assumes that the final focus spot is Gaussian,which is justified by calculating the spot size for a radial velocityfield balanced against diffusion¹³, then the spot will extend all theway out to the edge of the gel. Here diffusion can drive targets off thegel where there is no restoring focusing force and an applied DC biaswill sweep targets away from the gel where they will be lost. In thismanner the losses for ssSCODA can scale with the amount of time oneapplies a wash field; however the images used to generate FIGS. 13A-13Dindicate that in that example the focus spot has a full width halfmaximum (FWHM) of 300 μm and under bias it sits at approximately 1.0 mmfrom the gel centre. If it is assumed that there is 10 fmol of target inthe focus spot, then the concentration at the edge of the gel where abias is applied is 1e-352 M; there are essentially zero target moleculespresent at the edges of the gel where they can be lost under DC bias.This implies that the rate at which losses accumulate due to an appliedbias (i.e. washing step) is essentially zero. Although the desiredtarget may be lost from the system in other ways, for example byadsorbing to the sample well prior to injection, running off the edge ofthe gel during injection, re-annealing before or during focusing (in thecase of double stranded target molecules), or during extraction, all ofthese losses are decoupled from the purity of the purified target.

Aspects of the exemplary embodiments and examples described above may becombined in various combinations and subcombinations to yield furtherembodiments of the invention. To the extent that aspects of theexemplary embodiments and examples described above are not mutuallyexclusive, it is intended that all such combinations and subcombinationsare within the scope of the present invention. It will be apparent tothose of skill in the art that embodiments of the present inventioninclude a number of aspects. Accordingly, the scope of the claims shouldnot be limited by the preferred embodiments set forth in the descriptionand examples, but should be given the broadest interpretation consistentwith the description as a whole.

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1.-65. (canceled)
 66. A method of separating differentially methylated nucleic acid molecules, comprising: providing first and second nucleic acid molecules, the first and second nucleic acids molecules being at least 95% identical in sequence but being differentially methylated, contacting a matrix comprising an oligonucleotide probe with the first and second nucleic acids; and applying a time-varying electric field to the matrix while synchronously varying the mobility of the first and second nucleic acids, thereby separating the first and second nucleic acid molecules.
 67. The method of claim 66, further comprising collecting the first nucleic acid molecule from the matrix.
 68. The method of claim 66, further comprising collecting the second nucleic acid molecule from the matrix.
 69. The method of claim 66, wherein the time-varying electric field varies the mobility of the first and second nucleic acids.
 70. The method of claim 69, wherein the time-varying electric field varies the mobility by causing Joule heating within the matrix.
 71. The method of claim 66, wherein the difference between the binding energy of the first nucleic acid molecule to the oligonucleotide probe and the binding energy of the second nucleic acid molecule to the oligonucleotide probe is less than about 4 kcal/mol.
 72. The method of claim 66, wherein the difference between the melting temperature of the first nucleic acid molecule complexed with the oligonucleotide probe and the melting temperature of the second nucleic acid molecule complexed with the oligonucleotide probe is less than about 10° C.
 73. The method of claim 72, wherein the difference in temperatures is less than about 3° C.
 74. The method of claim 66, wherein the first and second nucleic acid molecules are between 100 and 1000 nucleotides in length.
 75. The method of claim 66, wherein the first and second nucleic acid molecules are less than 100 nucleotides in length.
 76. The method of claim 66, wherein the first nucleic acid molecule and the second nucleic acid molecule are methylated at different nucleotides.
 77. The method of claim 66, wherein the first nucleic acid molecule is hypermethylated relative to the second nucleic acid molecule.
 78. The method of claim 66, wherein the oligonucleotide probe is complimentary to at least a portion of the first and second nucleic acid molecules.
 79. The method of claim 66, wherein the first nucleic acid molecule originates from a fetus, the second nucleic acid molecule originates from a mother of the fetus and both the first and second nucleic acids are obtained from a blood sample from the mother.
 80. The method of claim 66, wherein both the first and the second nucleic acids comprise a gene implicated in cancer.
 81. The method of claim 66, further comprising amplifying or sequencing the first or the second nucleic acid.
 82. The method of claim 66, wherein the matrix comprises a plurality of different oligonucleotide probes.
 83. The method of claim 82, wherein a first portion of the plurality of different oligonucleotide probes is complimentary to the first nucleic acid molecule and a second portion of the plurality of different oligonucleotide probes is complementary to a third nucleic acid molecule, and wherein the method additionally comprises separating the third nucleic acid molecule from a fourth nucleic acid molecule.
 84. The method of claim 83, further comprising separating the first nucleic acid molecule from the third nucleic acid molecule.
 85. The method of claim 66, wherein the first and second nucleic acids molecules are at least 98% identical in sequence. 